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Optimization and Validation of a GC–FID Method for the Determination of Acetone-Butanol-Ethanol Fermentation Products

Xiaoqing Lin,

State Cardinal Laboratory of Materials-Oriented Chemical Engineering science, Nanjing, China

College of Biotechnology and Pharmaceutical Engineering, Nanjing Academy of Technology, Nanjing, People's republic of china

National Engineering Technique Enquiry Eye for Biotechnology, Nanjing, Communist china

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Abstruse

An improved, simple gas chromatography–flame ionization detection (GC–FID) method was developed for measuring the products of acetone-butanol-ethanol (ABE) fermentation and the combined fermentation/separation processes. The analysis fourth dimension per sample was reduced to less than 10 min compared to those of a conventional GC–FID (more than 20 min). The behavior of the compounds in temperature-programmed gas chromatographic runs was predicted using thermodynamic parameters derived from isothermal runs. The optimum temperature programming status was achieved when the resolution for each peak met the analytical requirement and the analysis time was shortest. With the exception of acetic acrid, the detection limits of the presented method for various products were below ten mg/Fifty. The repeatability and intermediate precision of the method were less than 10% (relative standard departure). Validation and quantification results demonstrated that this method is a sensitive, reliable and fast alternative for conventional investigation of the adsorption-coupled ABE fermentation process.

Introduction

Butanol is an important commercial chemical that is widely used in the plastic and material manufacture. By the 1940s, butanol fermentation, besides called acetone-butanol-ethanol (ABE) fermentation, was the second well-nigh of import industrial fermentation process next to ethanol fermentation. A rise in the chemical synthesis of butanol led to a decline in ABE fermentation in the 1960s (1). Still, due to its higher energy density than ethanol and because it can be used directly as a gasoline substitute in internal combustion engines, fermentation-derived butanol production on an industrial scale has attracted the interest of numerous companies (2). A typical feature of the solvent-producing Clostridium species is the transition from the acidogenic to the solventogenic phase. To obtain a holistic view of ABE fermentation and to maximize butanol productivity, belittling methods are required to measure acetic acid, butyric acrid (acidogenic stage), acetone, butanol and ethanol (solventogenic phase) (3).

Currently, diverse methods have been adult to measure the substrate and products of ABE fermentation. Loftier-performance liquid chromatography (HPLC) has been used to analyze both fermentation products and substrates (3, four). However, due to the poor resolution of butyric acid-acetone and acetone-ethanol in HPLC systems, information technology is of limited utilize. Buday et al. (three) described an improved belittling method for ABE fermentation, which involved adjusting the temperature of the Bio-Rad Aminex HPX87H column to xiv°C. The resolution between acetone and ethanol was also increased to 1.2, which allowed the quantitation of the concentrations of acetone and ethanol; however, the resolution between butyric acid and acetone was not significantly improved. Quantification of the solvent fermentation products based on gas chromatography with a flame ionization detector (GC–FID) has also been reported in the literature. In GC, different columns, such equally a glass cavalcade (10% CW-20M, 0.01% H_threePO₄, support fourscore/100 Chromosorb WAW) (five), a DB-WAX capillary column (30 m × 0.32 mm × 0.50 µm) (6) or an INNOWAX capillary column (15 1000 × 0.53 mm) (7), accept commonly been practical to determine the concentrations of acetone, butanol and ethanol. All the same, there are few manufactures about the GC method for the determination of ABE fermentation goop. The analysis time was always more than 20 min, and the details on the performance of these methods are not generally given in the literature, especially the repeatability, limit of detection (LOD), chromatogram and analysis times. Co-ordinate to Mes-Hartree and Saddler (8) and Doremus et al. (9), the major problem with several of the GC methods was the absorptive tailing of acerb acid, which can event in poor quantitation. Hence, as presented by Tsuey et al. (4), GC was used to analyze solvents, whereas HPLC was used to clarify organic acids. Although an improved GC method and a chromatogram were included in a later work, the assay time reached 19.28 min to avert the overlapping peaks that occurred between acetone and ethanol (iv).

An improved GC method for the assay and quantification of the culture broth with no sample pretreatment prior to GC assay is described; the method also includes a lower limit of detection, less time and a wider linear range. This method can also be practical in the adsorption-coupled ABE fermentation process, which tin heighten the fermentation rates and reactor productivity past adsorption resin. Various low boiling betoken eluents such as methanol were required to desorb the products from the different types of adsorbent. Information technology is necessary for each eluent to meliorate the method to accomplish fast and efficient analysis. In this newspaper, appropriate offline optimization models were conducted to overcome problems such as the curt analysis time and height overlapping effect without loss of resolution in the analysis of in situ product recovery (ISPR). For varied eluents, rapid and effective aligning tin be made to attain an effective method and shorten the fourth dimension. In the prediction process, acetic acid and butyric acid take obvious tailing and poor superlative shapes in isothermal runs, leading to the trouble in prediction of the superlative widths at one-half-pinnacle. According to the inquiry of Lu et al. (10), the relationship between elevation width and retention fourth dimension is suitable for peaks eluted at different temperatures. Hence, the peak widths of acids obtained from different linear temperatures were used in the prediction of the acme widths of acids during temperature programming. The predicted results concord with the experimental results. This improved method was applied to the monitoring and control of butanol fermentation (the glucose concentration was determined with a glucose analyzer).

Materials and Methods

Chemicals and reagents

HPLC-grade acetone, ethanol, methanol, butanol, acetic acid and butyric acid were purchased from Sinopharm Chemical Reagent Co. Stock solutions of acetone, ethanol, butanol, acetic acrid and butyric acid were prepared at concentrations of threescore, 40, 60, twenty and 20 g/Fifty, respectively, in distilled water. A series of solutions of each analyte was prepared with isobutanol every bit internal standard (IS; 6 g/50) for the structure of calibration curves.

Chromatographic conditions

The experiments were performed using a GC system (Agilent 7890, Santa Clara, CA) equipped with an FID. Separation of compounds was conducted on a 60 m HP-INNOWAX capillary column of 0.25 mm i.d., coated with polyethylene glycol (0.25 µm moving-picture show thickness), using nitrogen as the carrier gas. The injection volume was 1 µL and the flow rate was 2 mL/min. The injector temperature was 180°C with a carve up ratio of 90:1 and the FID temperature was 220°C. The oven temperature was programmed as follows: the column was held initially at seventy°C for 0.five min, so increased to 190°C at 20°C/min and held for iv min. Chromatographic data were recorded and integrated using Agilent Chemstation software.

The P2 medium was a synthetic medium and the concentrations of inorganic salts were very low. Equally the sample was injected into the GC, the sample first underwent gasification in the injection port liner, so entered the capillary column with a split ratio of 90:ane. The actual injection volume was 0.011 µL, so in that location was no column impairment. However, the injection port liner was contaminated later continuous use for more than iii months. The injection port liner was cleaned using acetone by an ultrasonic cleaner and new glass cotton was placed.

Microorganism and fermentation weather

The strain Clostridium acetobutylicum B3 was provided past the National Engineering science Technique Research Center for Biotechnology (Nanjing, Prc). The strain was maintained in a glycerol stock civilisation in a cryogenic freezer at –70°C. The seed medium consisted of the post-obit components (grand/L): soluble starch, 10; yeast excerpt, 3; peptone, 5; ammonium acetate, ii; sodium chloride, two; KH₂PO₄, 1; Thousand₂HPO₄, 1; MgSO₄, 3; FeSO₄ · 7H_iiO, 0.01. The P2 medium, with glucose as the carbon source, was sterilized at 121°C for 20 min in the l L stirred tank fermenter. The fermentation goop samples were centrifuged (Centrifuge 5804R, Eppendorf, Hamburg, Germany) at 13,000 grand at four°C for 3 min to separate sediments and the clear liquid was analyzed for ABE fermentation products. Before injection into the GC instrument, antiseptic samples and standards were filtered through 0.45 µm Whatman nylon filter (Gelman Science, Ann Arbor, MI) to remove insoluble materials that could block the cavalcade. All clear filtrate samples were kept frozen in sealed vials to maintain the stability of volatile components until they could be analyzed. Chromatographic samples were prepared with isobutanol as the IS (half-dozen g/L) in 2 mL screw-cap septum vials, which were then loaded into the autosampler. The growth of Clostridium acetobutylicum B3 was determined by the measurement of the optical density at 660 nm (OD660) with the use of a spectrophotometer.

Theory

Interpretation of memory times

To achieve adequate resolution in the shortest possible analysis fourth dimension, some theoretical and computational procedures were used to predict retentivity times and superlative widths of all analytes. In capillary GC, the chapters cistron value is influenced by thermodynamic parameters through the following equation:

(1)

where k is the capacity factor of the analyte i, R is the universal gas constant, T is the absolute temperature, β is the column phase ratio and ΔH and ΔDue south are the enthalpy and entropy of vaporization of analyte i from the stationary phase to the carrier gas phase, respectively. The variables ΔH and ΔS can exist causeless to be temperature contained and depend merely on the solute–solvent interactions (11). A linear human relationship between lnk and ane/T can be represented simply as

(2)

The bones equation of the retention time in the capillary column under isothermal conditions is given past

(iii)

where t _m is the column expressionless fourth dimension, a weak function of temperature. Information technology can be considered to be a constant at abiding menses rate fashion and is non influenced by oven temperature (x). Here, the t _{one thousand} value was determined by Agilent Chemstation software.

During a time interval Δt, the chemical compound will motion inside the column of a Δl length (12):

(iv)

when in a linear gradient temperature step

(5)

where α is the heating rate and 50 is the column length:

(6)

Hence, the temperature-programmed retention fourth dimension tin subsequently exist determined by assuming that the linear velocity of the carrier gas and the capacity factor are constant within each segment.

Estimation of peak widths

Autonomously from retention time, it is necessary to predict pinnacle width at half height for a programmed temperature run. Several methods based on the relationship in Eq. (7) were proposed for the adding of w _ane/2:

(7)

Get-go method for predicting superlative widths

The width of the zone for an increment of the column can exist described by Eq. (viii) by making the post-obit assumptions (13): (i) the plate height is considered to be a constant throughout the capillary column; (2) the effect of temperature on the diffusion characteristics is neglected; (iii) the zone broadening affected past the decompression is neglected; (iv) the thermal expansion of the carrier gas is neglected:

(8)

The value of the peak width, w _ane/2, in capillary GC is related to the actual zone width, W _one/2, and the velocity in the column u past the equation:

(9)

On the footing of Eqs. (7) and (viii), the zone width at the end of the column tin be expressed as

(10)

The value of w is obtained past the following equation:

(11)

where u _north is the velocity of the solute eluted isothermally at the retentiveness temperature T _r.

Second method for predicting peak widths

Due to the poor peak shapes of acerb acid and butyric acid in isothermal processes, another fashion of approaching the prediction of programmed temperature widths is described.

It has been proved that the relationship described by Eq. (7) in isothermal processes is compatible with that between the peak width in temperature-programmed GC and the invented retention time (14, fifteen). Thus, the pinnacle width is related to the invented retentiveness time, t _r ^*, which is the function of the retention temperature of component i T _ri. In other words, it is possible to approximate the elevation width in temperature-programmed GC to that in isothermal GC by means of the concept of invented retention time:

(12)

Results and Discussion

Prediction and optimization of GC separation

Information technology is possible to obtain the coefficients A and B from a series of isothermal data using Eq. (2). The retentivity fourth dimension values for acetone, methanol, ethanol, isobutanol and butanol were measured at 80, 110 and 140°C, whereas the residue were determined at 160, 180 and 220°C. Here, methanol was chosen equally an eluent to desorb the fermentation products from resin because of its low boiling point and because it was hands recoverable (16). The concentration of total products was estimated by the concentration of products in the fermentation liquid phase and those adsorbed in the resin. TableI shows that good linearity occurred between ln one thousand and one/T.

Tabular array I

Bones Data of Compounds in Isothermal Processes

Chemical compound	lnk = A + B/T		R ^two	w _1/2 = a + bt _r		R ²
Chemical compound	A	B × 10⁻³	R ^two	a × 10^two	b × 10²	R ²
Acetone	–10.41	3.35	0.9988	1.65	0.44	0.9999
Methanol	–eleven.32	3.82	0.9997	5.97	0.73	0.9999
Ethanol	–eleven.32	3.89	0.9999	5.44	0.46	0.9950
Isobutanol	–12.13	4.51	ane.0000	2.03	0.54	0.9995
Butanol	–12.23	four.66	0.9999	two.28	0.61	0.9984
Acetic acrid	–13.46	five.83	0.9998	–three.39	1.78	0.9997
Butyric acid	–13.41	half dozen.xi	1.0000	–one.92	1.xi	0.9995

Chemical compound	lnk = A + B/T		R ²	w _one/2 = a + bt _r		R ⁱⁱ
Chemical compound	A	B × 10^−three	R ²	a × ten²	b × 10²	R ⁱⁱ
Acetone	–10.41	3.35	0.9988	ane.65	0.44	0.9999
Methanol	–11.32	3.82	0.9997	5.97	0.73	0.9999
Ethanol	–11.32	3.89	0.9999	v.44	0.46	0.9950
Isobutanol	–12.thirteen	4.51	1.0000	2.03	0.54	0.9995
Butanol	–12.23	4.66	0.9999	2.28	0.61	0.9984
Acetic acid	–13.46	5.83	0.9998	–3.39	1.78	0.9997
Butyric acid	–13.41	6.11	one.0000	–one.92	1.eleven	0.9995

Table I

Basic Data of Compounds in Isothermal Processes

Compound	lnthousand = A + B/T		R ²	due west _i/two = a + bt _r		R ²
Compound	A	B × 10⁻³	R ²	a × 10²	b × 10²	R ²
Acetone	–ten.41	3.35	0.9988	1.65	0.44	0.9999
Methanol	–11.32	iii.82	0.9997	five.97	0.73	0.9999
Ethanol	–xi.32	3.89	0.9999	5.44	0.46	0.9950
Isobutanol	–12.13	4.51	ane.0000	2.03	0.54	0.9995
Butanol	–12.23	4.66	0.9999	2.28	0.61	0.9984
Acetic acid	–13.46	5.83	0.9998	–iii.39	1.78	0.9997
Butyric acid	–13.41	6.eleven	1.0000	–1.92	1.11	0.9995

Compound	lnm = A + B/T		R ²	due west _1/ii = a + bt _r		R ²
Compound	A	B × 10⁻ⁱⁱⁱ	R ²	a × 10ⁱⁱ	b × 10²	R ²
Acetone	–10.41	3.35	0.9988	1.65	0.44	0.9999
Methanol	–11.32	3.82	0.9997	5.97	0.73	0.9999
Ethanol	–eleven.32	3.89	0.9999	5.44	0.46	0.9950
Isobutanol	–12.13	four.51	1.0000	2.03	0.54	0.9995
Butanol	–12.23	4.66	0.9999	2.28	0.61	0.9984
Acetic acid	–13.46	five.83	0.9998	–3.39	1.78	0.9997
Butyric acid	–xiii.41	6.11	1.0000	–1.92	1.11	0.9995

Due to doubt in the determination of the peak widths of acetic acid and butyric acid in the isothermal processes, the coefficients a and b of these acids were obtained by experiments conducted at an initial temperature of lxx°C, increased at a rate of 5, x and 15°C/min; the others were determined from the isothermal data measured at lxxx, 110 and 140°C (Tabular arrayI).

Using the coefficients from TableI, the retention times, t _r, calculated as shown previously and summit widths at half height, w _1/two, were calculated past the method described previously. The 2d method significantly overestimated the peak widths. Hence, in addition to acetic acid and butyric acid, the peak widths shown in TableII were calculated by the method described previously.

Tabular array II

Comparison of Predicted and Experimental Retention Parameters

Compound	Retention time (min)			Summit width (s)
Compound	Experimental	Predicted	E _pred ^* × 10ⁱⁱ	Experimental × 10²	Predicted1 × 10²	Predicted2 × 10^two	E _pred1 × x³	E _predtwo × 10²
Acetone	3.760	iii.750	1.0	two.64	ii.80	3.08	i.threescore	0.44
Methanol	4.040	4.038	i.2	7.20	vi.96	8.38	two.40	one.xviii
Ethanol	4.204	4.204	0.0	4.66	5.52	6.97	8.60	two.31
Isobutanol	5.158	5.126	iii.2	2.87	3.09	3.88	ii.20	ane.01
Butanol	5.543	v.521	2.2	two.77	3.44	four.40	6.70	1.63
Acetic acrid	8.093	8.062	3.1	three.50		4.05		0.55
Butyric acrid	9.782	ix.669	11.three	3.48		four.00		0.52

Compound	Retentiveness time (min)			Top width (s)
Compound	Experimental	Predicted	E _pred ^* × 10²	Experimental × 10²	Predicted1 × 10²	Predicted2 × x^two	E _predi × 10³	E _pred2 × 10ⁱⁱ
Acetone	3.760	iii.750	1.0	2.64	two.80	3.08	1.sixty	0.44
Methanol	4.040	4.038	1.2	seven.20	half dozen.96	8.38	ii.40	1.eighteen
Ethanol	4.204	four.204	0.0	4.66	5.52	6.97	8.60	ii.31
Isobutanol	5.158	five.126	three.2	ii.87	3.09	3.88	ii.20	1.01
Butanol	five.543	5.521	2.2	2.77	three.44	four.40	6.lxx	i.63
Acetic acid	eight.093	8.062	3.1	3.50		4.05		0.55
Butyric acid	9.782	ix.669	xi.3	3.48		four.00		0.52

*East _pred = value (Experimental) – value (Predicted).

Table II

Comparison of Predicted and Experimental Retention Parameters

Chemical compound	Retention time (min)			Peak width (s)
Chemical compound	Experimental	Predicted	E _pred ^* × 10²	Experimental × 10²	Predicted1 × xⁱⁱ	Predicted2 × 10²	E _pred1 × 10³	E _pred2 × 10ⁱⁱ
Acetone	3.760	3.750	1.0	2.64	2.80	3.08	i.60	0.44
Methanol	4.040	4.038	i.2	7.20	6.96	viii.38	2.xl	1.18
Ethanol	4.204	4.204	0.0	iv.66	five.52	6.97	8.60	2.31
Isobutanol	5.158	5.126	iii.2	ii.87	3.09	3.88	2.20	1.01
Butanol	v.543	5.521	ii.two	2.77	3.44	4.xl	half dozen.70	ane.63
Acerb acid	eight.093	eight.062	iii.1	3.50		4.05		0.55
Butyric acid	9.782	9.669	eleven.three	3.48		4.00		0.52

Chemical compound	Memory time (min)			Pinnacle width (southward)
Chemical compound	Experimental	Predicted	E _pred ^* × ten²	Experimental × xⁱⁱ	Predicted1 × 10²	Predicted2 × x^two	East _predone × tenⁱⁱⁱ	E _predii × 10²
Acetone	3.760	three.750	1.0	ii.64	two.80	3.08	1.60	0.44
Methanol	four.040	4.038	i.2	7.twenty	6.96	8.38	two.40	i.eighteen
Ethanol	4.204	four.204	0.0	four.66	five.52	6.97	8.60	2.31
Isobutanol	5.158	5.126	3.two	two.87	3.09	three.88	2.20	i.01
Butanol	v.543	v.521	2.two	2.77	3.44	iv.40	half-dozen.lxx	i.63
Acetic acid	8.093	eight.062	3.one	3.50		four.05		0.55
Butyric acrid	9.782	9.669	11.3	three.48		4.00		0.52

*E _pred = value (Experimental) – value (Predicted).

Considering the boiling point of acetone (the showtime eluted top) and the resolution between acetone and methanol, the initial oven temperature was set at 70°C when the retention gene was 0.five. The last oven temperature was optimized and set at 190°C to obtain adequate LODs and loss of the stationary phase of the GC column. Resolution was measured by Eq. (xiii) to describe how well compounds were separated:

(thirteen)

To minimize the analysis time, 1.5 was chosen as the required resolution, R _req, which was sufficient for utmost accurateness (17). The maximum allowed heating rate was 45°C/min, which was used as the initial heating charge per unit in the prediction of peak resolution. The terminal heating rate was identified past decreasing the maximum allowed heating rate to meet the required peak resolution. A temperature programming rate above 20°C/min resulted in partial co-elution of methanol with ethanol (R < 1.3). The agree of 0.v min at the initial temperature allowed consummate separation of the analyte peaks and reduced the assay time.

The flow chart of the proposed optimization algorithm for ABE fermentation products is presented in the Supplementary materials. The predicted and experimental retention times and elevation widths at one-half-heights of all compounds are shown in Table2. Excellent agreement was found between the predicted and experimental retention parameters. Except for the acme widths of acids, which were predicted by the previously described second procedure, the kickoff process gave significantly meliorate peak width predictions than the second procedure. The reason for this is partly because the linear velocity of a elevation'due south may change chop-chop nether temperature programming.

The applicability of the proposed method was tested by analyzing three samples. Figure1 shows the chromatograms obtained from the analysis of model fermentation broth (Figure1A), ABE fermentation broth (Figure1B) and model eluent and the mixture of methanol and model fermentation goop, diluted with distilled water (Effigy1C). All compounds were clearly identified with no significant interferences from the sample matrix.

Figure 1.

GC chromatograms obtained from the analysis of reference standards in: model fermentation goop sample (A); fermentation broth sample (B); model eluent (C) sample. Peaks: one, acetone; 2, methanol; 3, ethanol; iv, butanol; v, acetic acid; 6, butyric acid; IS, isobutanol.

Method validation

Calibration curve and linearity

Calibration curves were prepared past plotting the height expanse ratios (elevation area of analytes/peak area of IS) versus concentration. As shown in TableIII, adept linearity for the method was obtained, with correlation coefficients in the range of 0.9992–0.9997, by using a serial of solutions containing various concentrations of ABE, acetic acid and butyric acid. The linear ranges of acetone, butanol, ethanol, acerb acid and butyric acid were 0.3–30, 0.2–xx, 0.iii–30, 0.i–10 and 0.1–10, respectively. Compared with the results reported in the previous related newspaper (the linear ranges of acetone, butanol, ethanol, acetic acid and butyric acrid were 0–8, 0–four, 0–4, 0–iv and 0–eight g/L, respectively) (three), this optimized method has wider linear range.

Table III

Features of the Proposed GC Methods for the Determination of Acetone, Ethanol, Butanol, Acerb Acid and Butyric Acrid

Analyte	Gradient × 10² ± SD × 10^iv	Intercept × 10² ± SD × 10⁴	Linear range (chiliad/Fifty)	Due north	R ²	LOD (mg/L)
Acetone	12.33 ± i.0	–2.34 ± ii.1	0.3–thirty	7	0.9997	three.i
Ethanol	11.94 ± 9.8	–ane.39 ± 31	0.two–20	vii	0.9998	4.vii
Butanol	sixteen.87 ± 1.1	–2.28 ± 2.9	0.iii–thirty	seven	0.9998	two.9
Acetic acrid	5.60 ± vi.1	–1.46 ± 9.0	0.1–x	7	0.9997	18.0
Butyric acid	11.85 ± 10	–1.40 ± 14	0.one–10	7	0.9992	8.3

Analyte	Gradient × 10² ± SD × 10^four	Intercept × 10² ± SD × x⁴	Linear range (g/L)	North	R ²	LOD (mg/50)
Acetone	12.33 ± i.0	–2.34 ± 2.1	0.three–30	7	0.9997	three.1
Ethanol	eleven.94 ± nine.viii	–1.39 ± 31	0.2–20	7	0.9998	4.7
Butanol	sixteen.87 ± 1.ane	–2.28 ± 2.ix	0.3–xxx	7	0.9998	ii.nine
Acetic acrid	5.60 ± half-dozen.1	–i.46 ± 9.0	0.1–x	seven	0.9997	eighteen.0
Butyric acid	eleven.85 ± ten	–1.40 ± 14	0.one–ten	7	0.9992	8.3

Table Iii

Features of the Proposed GC Methods for the Determination of Acetone, Ethanol, Butanol, Acerb Acid and Butyric Acid

Analyte	Slope × 10² ± SD × 10^iv	Intercept × 10² ± SD × x⁴	Linear range (g/L)	Northward	R ⁱⁱ	LOD (mg/L)
Acetone	12.33 ± 1.0	–two.34 ± 2.1	0.three–thirty	7	0.9997	3.1
Ethanol	11.94 ± 9.8	–one.39 ± 31	0.2–20	7	0.9998	four.7
Butanol	xvi.87 ± 1.1	–2.28 ± 2.9	0.3–30	seven	0.9998	2.9
Acetic acid	5.60 ± 6.1	–1.46 ± nine.0	0.i–10	7	0.9997	18.0
Butyric acrid	11.85 ± 10	–one.40 ± fourteen	0.1–10	7	0.9992	eight.iii

Analyte	Slope × ten² ± SD × x⁴	Intercept × ten² ± SD × 10^four	Linear range (k/Fifty)	N	R ²	LOD (mg/L)
Acetone	12.33 ± 1.0	–ii.34 ± 2.1	0.iii–30	7	0.9997	three.1
Ethanol	11.94 ± 9.viii	–1.39 ± 31	0.two–20	7	0.9998	4.7
Butanol	16.87 ± ane.ane	–ii.28 ± 2.9	0.iii–thirty	7	0.9998	ii.9
Acetic acrid	v.lx ± 6.ane	–1.46 ± 9.0	0.1–x	seven	0.9997	eighteen.0
Butyric acid	11.85 ± 10	–1.40 ± 14	0.1–ten	seven	0.9992	eight.three

Limits of detection

LODs were evaluated from a betoken to noise ratio (Due south/North) equal to iii. Equally listed in Tabular arrayIII, the method allowed the detection of ABE in the range of 2.9–4.7 mg/L, whereas the assigned values were xviii and viii.3 mg/L for acerb acrid and butyric acid, respectively. Compared with the results reported in the previous related paper (LOD ranges of acetone, butanol, ethanol, acetic acid and butyric acrid were 100, 80, fourscore, xc and 100 mg/L, respectively) (3), this optimized method provided lower detection limits of the analytes.

Recovery

The recoveries of this method were quantified at three concentration levels, which are shown in TableIV. The recoveries of the five analytes ranged from 100 to 113%. Analysis of variance was performed, which showed no statistically significant divergence between the recoveries with respect to concentration. In conclusion, these recoveries were consistent, precise and reproducible in the same samples under unlike concentrations.

Table Iv

Recovery of Compounds in Fermentation Broth Determined at Three Concentration Levels

Compounds	Concentration in fermentation broth (grand/L)	Added (g/L)	Recovery (%)	RSD (%) (n = 3)
Acetone	two.909	2.156	104.1	0.40
		ii.934	106.5	0.43
		four.028	104.ix	0.35
Ethanol	0.433	0.362	103.vii	1.45
		0.624	112.9	1.88
		0.996	110.1	1.22
Butanol	6.608	3.864	102.1	1.05
		5.980	102.0	0.65
		7.198	100.8	0.75
Acerb acrid	1.224	0.466	112.2	0.62
		0.896	112.4	one.50
		one.660	110.4	1.74
Butyric acrid	1.336	0.646	105.five	0.53
		ane.050	106.six	2.xi
		1.656	106.7	i.24

Compounds	Concentration in fermentation broth (g/L)	Added (chiliad/L)	Recovery (%)	RSD (%) (n = 3)
Acetone	2.909	two.156	104.one	0.40
		ii.934	106.5	0.43
		4.028	104.9	0.35
Ethanol	0.433	0.362	103.7	1.45
		0.624	112.ix	1.88
		0.996	110.1	1.22
Butanol	6.608	3.864	102.one	i.05
		5.980	102.0	0.65
		7.198	100.8	0.75
Acetic acid	1.224	0.466	112.2	0.62
		0.896	112.4	1.fifty
		one.660	110.4	one.74
Butyric acid	1.336	0.646	105.5	0.53
		ane.050	106.half-dozen	2.11
		ane.656	106.vii	ane.24

Table IV

Recovery of Compounds in Fermentation Broth Determined at Iii Concentration Levels

Compounds	Concentration in fermentation broth (g/L)	Added (k/L)	Recovery (%)	RSD (%) (northward = 3)
Acetone	2.909	2.156	104.1	0.40
		two.934	106.5	0.43
		4.028	104.9	0.35
Ethanol	0.433	0.362	103.7	ane.45
		0.624	112.9	1.88
		0.996	110.1	ane.22
Butanol	half dozen.608	iii.864	102.i	1.05
		5.980	102.0	0.65
		7.198	100.eight	0.75
Acetic acid	one.224	0.466	112.ii	0.62
		0.896	112.4	1.fifty
		ane.660	110.4	1.74
Butyric acid	1.336	0.646	105.v	0.53
		1.050	106.half dozen	2.11
		1.656	106.7	ane.24

Compounds	Concentration in fermentation broth (grand/Fifty)	Added (g/L)	Recovery (%)	RSD (%) (n = 3)
Acetone	2.909	2.156	104.1	0.40
		2.934	106.5	0.43
		iv.028	104.nine	0.35
Ethanol	0.433	0.362	103.7	1.45
		0.624	112.nine	i.88
		0.996	110.1	one.22
Butanol	half-dozen.608	3.864	102.1	1.05
		5.980	102.0	0.65
		vii.198	100.8	0.75
Acetic acid	1.224	0.466	112.2	0.62
		0.896	112.4	1.50
		1.660	110.4	ane.74
Butyric acid	1.336	0.646	105.5	0.53
		1.050	106.six	ii.11
		i.656	106.7	1.24

Precision and repeatability

The precision of the method was adamant from multiple analyses of the same sample on unlike days. The inter-day precision was calculated from the fermentation goop at 25.5, 55 and 76 h (Figure2). The relative standard deviations (RSDs) were less than 10% at various concentrations of the fermentation broth. These results are shown in TableFive. The satisfactory precision indicated practiced performance and stability of the method for the quantitative analysis of the compounds in the ABE fermentation broth.

Figure 2.

Concentration profiles of the substrate and products from ABE fermentation. Glucose concentration was determined with glucose analyzer and products were measured using the described method. Glucose, white circles; butanol, squares; acetone, diamonds; ethanol, upside-down triangles; butyric acid, triangles; acetic acid, black circles (A); growth of Clostridium acetobutylicum B3 (OD660) from ABE fermentation (B).

Tabular array V

Intra-24-hour interval and Inter-Solar day Precision of the Proposed Method

Analyte	Concentration in fermentation broth	Intra-day assay	Inter-24-hour interval analysis (4 days)
	(g/Fifty)	RSD (%) (n = half dozen)	RSD (%) (n = 3)
Acetone	0.575	0.92	iv.10
	2.384	1.61	seven.44
	4.244	0.55	8.52
Ethanol	0.201	one.00	iii.96
	0.326	2.24	seven.57
	0.891	0.77	9.25
Butanol	1.554	0.x	i.26
	6.489	0.23	0.56
	ten.300	0.17	0.62
Acetic acid	i.658	one.60	four.73
	0.990	iii.55	5.87
	0.602	1.35	3.43
Butyric acid	two.595	i.72	three.92
	0.766	3.76	4.49
	0.221	ii.57	5.05

Analyte	Concentration in fermentation goop	Intra-day assay	Inter-day assay (4 days)
	(thou/L)	RSD (%) (n = 6)	RSD (%) (n = three)
Acetone	0.575	0.92	4.x
	2.384	ane.61	vii.44
	four.244	0.55	eight.52
Ethanol	0.201	ane.00	3.96
	0.326	2.24	7.57
	0.891	0.77	9.25
Butanol	one.554	0.x	ane.26
	6.489	0.23	0.56
	10.300	0.17	0.62
Acetic acrid	1.658	1.lx	four.73
	0.990	iii.55	5.87
	0.602	1.35	3.43
Butyric acid	2.595	1.72	three.92
	0.766	3.76	iv.49
	0.221	2.57	5.05

Table 5

Intra-Day and Inter-Day Precision of the Proposed Method

Analyte	Concentration in fermentation broth	Intra-twenty-four hours assay	Inter-day assay (four days)
	(g/L)	RSD (%) (n = six)	RSD (%) (north = 3)
Acetone	0.575	0.92	four.10
	ii.384	ane.61	vii.44
	4.244	0.55	8.52
Ethanol	0.201	1.00	3.96
	0.326	2.24	7.57
	0.891	0.77	9.25
Butanol	ane.554	0.ten	1.26
	6.489	0.23	0.56
	10.300	0.17	0.62
Acetic acid	one.658	1.60	4.73
	0.990	three.55	5.87
	0.602	1.35	iii.43
Butyric acid	2.595	ane.72	3.92
	0.766	3.76	4.49
	0.221	2.57	v.05

Analyte	Concentration in fermentation broth	Intra-solar day assay	Inter-day assay (4 days)
	(g/L)	RSD (%) (north = half dozen)	RSD (%) (n = iii)
Acetone	0.575	0.92	4.10
	two.384	1.61	seven.44
	iv.244	0.55	8.52
Ethanol	0.201	1.00	3.96
	0.326	2.24	7.57
	0.891	0.77	9.25
Butanol	1.554	0.10	i.26
	6.489	0.23	0.56
	10.300	0.17	0.62
Acetic acrid	one.658	i.60	4.73
	0.990	three.55	5.87
	0.602	1.35	iii.43
Butyric acid	ii.595	one.72	iii.92
	0.766	iii.76	4.49
	0.221	2.57	5.05

Measurement of fermentation samples

The command batch fermentation experiment was run at 37°C and pH 5 with 60 g/L of glucose in P2 medium. The fermentation samples were removed at fourth dimension intervals and candy immediately for chromatography. Effigytwo shows the product profiles, substrate uptake and OD660 of Clostridium acetobutylicum B3 over the form of 97.v h. ABE product by Clostridium acetobutylicum B3 in a batch culture was characterized by two distinct phases, the acidogenic stage (0–30 h) and the solventogenic phase (xxx–76 h). During the acidogenic phase, acerb and butyric acid were accumulated to concentrations of approximately 2.twenty and 2.64 g/L, respectively. The concentration of acids decreased speedily and ABEs were accumulated in the culture during the solventogenic phase. After 76 h of fermentation, the civilization produced four.24 g/L of acetone, 10.xxx g/50 of butanol and 1.15 m/L of ethanol, resulting in ABE productivity of 0.21 (g/L · h) and ABE yield of 0.27 grand/yard. The concentrations of acerb and butyric acid at the finish of fermentation were 0.59 and 0.22 g/L, respectively. Although the fermentation was run for 97.5 h, the culture stopped producing butanol at 76 h, leaving two.3 g/Fifty of rest glucose. The major reason for abeyance of the fermentation procedure was the hydrophobic nature of butanol (1, 18). It is toxic and its primary effects appear to be disrupting membrane-linked functions, decreasing intracellular ATP levels and inhibiting carbohydrate uptake (one, 18). In the batch fermentation process, the growth of Clostridium acetobutylicum B3 was non inhibited at a butanol concentration of 4.59 chiliad/L. The growth of Clostridium acetobutylicum B3 was 50% inhibited at a butanol concentration of 7.29 g/L, and so was totally inhibited at a butanol concentration of 10.34 g/L (Figureii).

Conclusions

The present paper demonstrates a unproblematic, sensitive, reliable and fast GC–FID method for measuring the components of the ABE fermentation broth (acetone, ethanol, butanol, acetic acrid and butyric acid). These features brand it a reliable and convenient tool for the analysis of the ABE fermentation process and fermentation coupled with separation. Using the developed model, the predicted values of retention times and peak widths agreed with the experimental values. The optimal chromatographic atmospheric condition can exist obtained by the predicted retention times and peak widths. Methanol was called as a suitable eluent for the characteristics of the absorbent used in this article. Other adsorbents were considered; a fast, reliable method for the direct analysis of the eluent of in situ adsorption in ABE fermentation may also be optimized quickly by the theory described previously in the paper.

Acknowledgments

This work was supported past Program for Changjiang Scholars and Innovative Research Team in University (Grant No.: IRT1066), National Outstanding Youth Foundation of China (Grant No.:21025625), National Loftier-Tech Research and Evolution Plan of Communist china (863 Program, 2012AA021200), Jiangsu Provincial Natural Science Foundation of Red china (Grant No.: SBK201150207) and Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Xiaoqing Lin was supported by the average higher graduate student inquiry innovative projects of Jiangsu province (CXZZ11_0362).

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Author notes

These authors equally contributed to this study.

How to Read Gc of 2-butanol Syntheses

Source: https://academic.oup.com/chromsci/article/52/3/264/275226

How to Read Gc of 2-butanol Syntheses

Commodity Contents

Optimization and Validation of a GC–FID Method for the Determination of Acetone-Butanol-Ethanol Fermentation Products

Abstruse

Introduction

Materials and Methods

Chemicals and reagents

Chromatographic conditions

Microorganism and fermentation weather

Theory

Interpretation of memory times

Estimation of peak widths

Get-go method for predicting superlative widths

Second method for predicting peak widths

Results and Discussion

Prediction and optimization of GC separation

Method validation

Calibration curve and linearity

Limits of detection

Recovery

Precision and repeatability

Measurement of fermentation samples

Conclusions

Acknowledgments

References

Author notes

Supplementary data

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