Glycerol determination in biodiesel and biodiesel blends

according to ASTM D 7591

J. Gandhi1 and A. Wille2
1
Metrohm USA Inc., Riverview FL 33578, USA
2
Metrohm International Headquarters, CH-9101 Herisau, Switzerland

The presented ion chromatographic method is applicable to all biodiesel types and
blends. Before chromatographic separation, free glycerol and bound glycerol are
isolated by a straightforward extraction and saponification-extraction technique. Pulsed
amperometric detection following chromatographic separation achieves an outstanding
method detection limit (MDL) of 0.5 ppm by mass for glycerol and thus easily fulfills
ASTM and EN performance specifications. The described method fully complies with
ASTM D 7591.

Biodiesel
The four primary driving forces behind biofuels are the world's increasing thirst for petroleum
(80 Mbarrels/day), the diminishing supply of fossil fuels, global warming, and the intention to
reduce the dependence on fuel imports. Additionally, most biofuels are produced by
straightforward manufacturing processes, are readily biodegradable and nontoxic, have low
emission profiles, and can be used as is or blended with conventional fuels.
Biodiesel is produced by transesterifying the triglycerides in the parent oil or fat with an
alcohol, usually methanol, in the presence of a catalyst (base, acid, or enzyme) to yield fatty
acid methyl esters (FAME) and free glycerol as coproduct (Figure 1). As reaction rates under
acid or enzyme catalysis are relatively slow, most producers use the rapid alkali-catalyzed
transesterification.

Figure 1: Base-catalyzed transesterification of a triglyceride with methanol

Incomplete reaction leads to the formation of residual glycerol intermediates such as mono-,
di- and triacylglycerides (bonded glycerols). In contrast, complete conversion results in the
formation of highly water-soluble glycerol (free glycerol). The latter is separated from the
final product at the end of the production process. However, traces of glycerol are frequently
found in the ester phase. Both free and bonded glycerols (= total glycerol) lead to severe
operational problems such as injector and valve deposits or filter clogging. Accordingly, the
US ASTM D 6751 [1] specifies a maximum total glycerol content of 2400 ppm (0.24%),
while the European EN 14214 [2] stipulates 2500 ppm (0.25%). In both standards, the free
glycerol content is limited to 200 ppm (0.02%).
Based on the analysis of biodiesel blends made from coconut oil, this article demonstrates
sensitive analysis of the free and total glycerol content via simple and innovative ion

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chromatography followed by pulsed amperometric detection (PAD) according to ASTM D
7591 [3].

Experimental
Instrumentation
The chromatography system consisted of the 850 Professional IC with Amperometric
Detector and the 858 Professional Sample Processor (all Metrohm AG, Figure 2). For all
separations, a Metrosep Carb 1 - 150/4.0 anion-exchange column was used with a flow rate of
1 mL/min. The injection valve was fitted with a 20 L injection loop and separation was
achieved by isocratic elution employing a 100 mmol/L NaOH eluent.
The amperometric detector consists of a gold working electrode in combination with a solid-
phase reference electrode and a stainless-steel auxiliary electrode. A triple-step potential
waveform was applied.
Instrument control, data acquisition, and processing were performed using MagIC Net
software (Metrohm).

Figure 2: 850 Professional IC with IC Amperometric Detector and 858 Professional Sample Processor

Reagents
Glycerol standard and potassium hydroxide were reagent grade and purchased from Sigma-
Aldrich (Milwaukee, WI, USA). Synthetic biodiesel blends B2 to B20 were produced by
respective mixing of biodiesel made from glycerol-containing coconut-oil and low-sulfur
petroleum diesel. All standard solutions and eluents were prepared from deionized water with
a specific resistance higher than 18 Mcm.

Extraction and saponification

While the extraction of free glycerol can be performed by a simple separating funnel,
saponification-extraction for bound glycerol requires a commercially available reflux system
capable of heating the reaction mixture to 90 C.

(a) Free glycerol

Generally, a high free glycerol content points to incomplete separation of the ester and
glycerol phase. Because of the high water solubility of the triol 1000 mg and more will
dissolve in a liter of water free glycerol can be readily extracted from biodiesel or biodiesel
blends.
The procedure comprises the addition of 45 g of distilled water to approximately 5 g of
sample. After vigorous shaking for five minutes the sample is allowed to stand for another

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five minutes. After phase separation, an aliquot of the aqueous phase is filled into a
chromatography vial and placed on the Sample Processor for analysis.

(b) Total glycerol

Total glycerol is the sum of free and bound glycerol. The latter is the sum of residual mono-,
di-, and triglycerides and stems from incomplete esterification reactions. Glycerides are
removed from the organic phase by saponification reaction with sodium hydroxide and
subsequent extraction of the generated glycerol with water.
In a reflux system, 20 mL of 0.01 mol/L potassium hydroxide is added to approximately 2 g
of sample. The mixture is heated to reflux for one hour. After cooling to room temperature,
the volume of the mixture is made up to 50 mL. The released glycerol is then extracted into
the aqueous phase according to the procedure described above.

Results
Calibration and method detection limit
Calibration standards range from 0.5 to 100 mg/L. Calibration is linear providing a correlation
coefficient of 0.99996 with a relative standard deviation better than 0.437%.
The detection limit was determined by a 15-fold injection of a 0.5 ppm glycerol standard. The
excellent MDL of 0.5 ppm glycerol by mass (0.7104%) exceeds by far the maximum free
glycerol content of 200 ppm (0.02%) required by the ASTM D 6751 or EN 14214.

Figure 3: Glycerol determination using pulsed amperometric detection.

Free and total glycerol content in different biodiesel blends and a coconut-oil biodiesel
sample
Whereas the total glycerol limit in ASTM D 6157 and EN 14214 is 0.24% and 0.25%,
respectively, the allowed maximum content of free glycerol is only 0.02% in both standards.
The pure biodiesel sample B100 contains 0.027% of free and 0.62% of total glycerol (Table
1) and thus exceeds the limits stipulated by the two standards. Before being used as fuel or
being blended with petroleum diesel, the coconut methyl ester first has to be freed of its
excess glyceride and glycerol contents.
According to Table 1 and Figure 4 the free and total glycerol contents in all investigated
biodiesel blends B2 to B20 are below 0.0051 and 0.124%.

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Figure 4: PAD chromatogram with (a) free and (b) total glycerol peaks in a B2 blend.

Figure 5: Stacked PAD chromatograms of different biodiesel blends (B2B20).

Table 1: Free and total glycerol content in different biodiesel blends

Free glycerol Total glycerol
Blend Statistic parameters Determined Expected Determined Expected
Mean value [%]weight, n = 3 0.000559 0.000539a 0.01086 0.012434a
B2 Standard deviation [%]weight 0.000003 0.000344
Relative standard deviation 0.446% 3.168%
Mean value [%]weight, n = 3 0.001322 0.001348a 0.029752 0.031084a
B5 Standard deviation [%]weight 0.000007 0.001263
Relative standard deviation 0.559% 4.246%
Mean value [%]weight, n = 3 0.002759 0.002695a 0.061032 0.062168a
B10 Standard deviation [%]weight 0.000321 0.000898
Relative standard deviation 1.162% 1.472%
Mean value [%]weight, n = 3 0.005046 0.005390a 0.124133 0.124336a
B20 Standard deviation [%]weight 0.000229 0.001802
Relative standard deviation 0.453% 1.452%
Mean value [%]weight, n = 6 0.026950 0.026950 0.621680 0.621680
B100 Standard deviation [%]weight 0.000570 0.019430
(Coconut oil) Relative standard deviation 0.021% 0.031%
a
calculated by applying the appropriate dilution factor

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 Conclusion
Free and bound glycerol is determined by ion chromatography and subsequent pulsed
amperometric detection in accordance with ASTM D 7591. It is a simple, cost-efficient, and
very accurate method that includes a straightforward extraction and saponification-extraction
for determining the free and bound glycerol content, respectively. With an MDL of 0.5 ppm
by mass for total glycerol, IC-PAD easily exceeds the requirements of ASTM and DIN
standards.

References
[1] ASTM D 6751, Standard specification for biodiesel fuel blend stock (B100) for middle
distillate fuels.
[2] DIN 14214, Automotive fuels fatty acid methyl esters (FAME) for diesel engines
requirements and test methods.
[3] ASTM D 7591, Standard test method for determination of free and total glycerin in
biodiesel blends by anion exchange chromatography.