Analysis of Glycerin and Glycerides in

Biodiesel (B100) Using ASTM D6584 and

EN14105

Application

HPI/Petrochemicals/Polymers

Author to replace, supplement, or extend traditional

petroleum fuels. Biodiesel is produced by a process
James D. McCurry called transesterification. The vegetable oil is
Agilent Technologies, Inc. reacted with methanol in the presence of a catalyst
2850 Centerville Road to produce a mixture of fatty acid methyl esters
Wilmington, DE 19808 (FAME) and glycerin. After removal of the glycerin
USA and other contaminants, the remaining FAME mix-
ture is pure biodiesel. Depending on the oil source,
Chun-Xiao Wang
a typical biodiesel contains FAME mixtures having
Agilent Technologies (Shanghai) Co., Ltd.
both saturated and unsaturated carbon chains
412 Ying Lun Road from C8 to C24. Table 1 shows the distribution and
Waigaoqiao Free Trade Zone relative amounts of FAME found in biodiesel made
Shanghai 200131 from common plant oils.[1]
Peoples Republic of China
Pure biodiesel is generally not used as a fuel, but
instead it is blended with petroleum diesel.
Abstract Biodiesel is defined by the notation Bxx, where xx
indicates the volume percent of FAME content in
The analysis of free glycerin (glycerol) and total glyc- the liquid. Using this nomenclature, B100 is pure
erides (mono-, di-, and triglycerides) in B100 biodiesel FAME, B50 contains 50 volume % FAME, B5 con-
was performed according to ASTM method D6584 and tains 5 volume % FAME, etc. Common commercial
CEN method EN14105. Method improvements were biodiesel blends are B2, B5, and B20.
demonstrated through the use of a 530-m id high-tem-
perature fused-silica retention gap coupled to the analyti- Before biodiesel can be sold as a fuel or blending
cal column. This was made possible with an Agilent stock, it must first meet a defined standard. ASTM
Capillary Flow Technology Ultimate Union designed for standard D6751 and European Committee of Stan-
inert, high-temperature GC oven operation. This configu- dardization (CEN) standard EN14214 set similar
ration on the Agilent 7890A GC System showed calibra- specifications for biodiesel blending and motor
tion and precision performance that exceeded both D6584 fuels.[2,3] In each standard, an important specifi-
and EN14105 specifications. This application provides cation is a limit on the amounts of free glycerin
complete system configuration as well as guidelines for and glycerides in biodiesel. Free glycerin is a
successful analysis of free glycerin and total glycerides in byproduct of biodiesel production. Mono-glyc-
biodiesel. erides, diglycerides, and triglycerides are partially
reacted oils that may be contaminants in the fin-
ished biodiesel. High amounts of free glycerin can
Introduction cause problems due to separation. High amounts of
glycerides and glycerin can result in increased
Biodiesel is a motor or heating fuel produced from
engine deposits. Table 2 shows the limits set by
renewable vegetable oils or animal fats. With the
each standard.
high cost and limited availability of crude oil,
renewable fuels like biodiesel are seen as a way
Table 1. Distribution and Relative Amounts of FAMEs Derived from Vegetable Oils
Weight Percent FAMEs

C20:0 C20:1
Oil type C8:0 C10:0 C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C22:0 C22:1
Rapeseed 25 0.2 12 1015 1020 510 0.9 5060
Soybean 0.3 711 01 36 2234 5060 210 510
Palm 16 3247 16 4052 211
Coconut 59 410 4552 1318 710 14 58 13
Palm kernel 24 37 4451 1419 69 01 13 1018 12 12

Table 2. Free and Total Glycerin Specifications for Biodiesel

EN14214 ASTM D6571

Limit (% m/m) Test method Limit (% m/m) Test method

Free glycerin 0.02 max EN14105 0.020 max D6584
Monoglycerides 0.80 max EN14105 NA D6584
Diglycerides 0.20 max EN14105 NA D6584
Triglycerides 0.20 max EN14105 NA D6584
Total glycerin 0.25 max EN14105 0.240 max D6584

ASTM and CEN have defined several physical and syringe. An Agilent Capillary Flow Technology Ulti-
chemical test methods to meet the standard speci- mate Union was used to join the retention gap and
fications. An important chemical test measures the the analytical column. Table 4 shows the GC oper-
free glycerin and glyceride content in B100. Two ating conditions used for this analysis.
gas chromatographic methods, EN14105 and
D6584, were developed to make this measure- Standard and Sample Preparation
ment.[4,5] Both are nearly identical in sample
preparation, instrument configuration, operating Agilent Technologies biodiesel standards were
conditions, and reporting. Since glycerin and glyc- used containing glycerin, monoolein, diolein, tri-
erides are polar and high boiling, they must first olein, butanetriol (internal standard #1), and tri-
be derivatized to improve volatility and reduce caprin (internal standard #2) at concentrations
activity before injection into the GC. A cool-on- specified in the ASTM and CEN methods. A list of
column inlet (COC) and high-temperature capillary these standards and other chemical reagents used
column are used to make the analysis of these for this analysis are shown in Table 3.
compounds easier. Another important considera-
Five GC calibration standards were prepared by
tion when using these methods is the source of the
mixing aliquots of the individual stock standards
biodiesel. Both methods were developed for B100
in proportions specified by the ASTM and CEN
derived from vegetable oils such as rapeseed, soy-
methods. After mixing, 100 L of the derivatization
bean, sunflower, and palm. It is known that these
agent, N-Methyl-N-(trimethylsilyl)trifluoroac-
methods are not suitable for B100 derived from
etamide (MSTFA) was added to each calibration
lauric acid oils, such as coconut and palm kernel
standard. After 20 minutes, 8 mL of reagent grade
oils.
n-heptane was added to each calibration standard.
These final reaction mixtures were directly
Experimental injected into the gas chromatograph.

Sample preparation followed the procedure in the

Instrument Configuration
ASTM and CEN methods. Two samples of B100,
Table 3 lists the details of the GC configuration from soybean oil and rapeseed oil, were used for
used for this work. A 530-m id high-temperature this application. Each sample was run two times
retention gap was used between the on-column over four consecutive days with fresh calibration
inlet and the analytical capillary column to standards prepared and run for each analysis.
improve sample vaporization and provide easy
sample injection using a standard tapered needle

2
Table 3. System Configuration (SP1 7890-0294)
Standard 7890A GC hardware
G3440A Agilent 7890A Series GC
Option 122 Cool-on-column inlet with electronic pneumat-
ics control (EPC)
Option 211 Capillary flame ionization detector (FID) with
EPC control
G2613A Agilent 7683 Autoinjector
Columns
Analytical column DB-5ht, 15 m x 0.32 mm id x 0.1-m film
(part no. 123-5711)
High-temperature retention gap Deactivated fused-silica tubing, 1 m x
0.53 mm id (part no.160-2865-5 comes in
5-m lengths)
Union Capillary Flow Technology Ultimate Union Kit
(part no. G3182-61580)
Union ferrules 0.32-mm column Siltite ferrules
(part no. 5188-5362)
0.53-mm column Siltite ferrules
(part no. 5188-5363)
Data system
Agilent Multitechnique ChemStation
Consumables
5181-1267 10-L Teflon fixed autoinjector syringe
Standards and reagents
5190-1408 Biodiesel D6584 kit, 5 calibration standard solu-
tions and 2 internal standard solutions
5190-1407 Biodiesel MSTFA derivatization kit, 10 x 1 mL
ampoule

Table 4. Instrument Conditions

Cool-on-column inlet
Mode Ramped
Initial temperature oven track, approx 50 C
Pressure 7.6 psi helium
Injection amount 1 L
Initial column flow 3.0 mL/min, constant pressure mode
FID temperature 380 C
Oven temperature program 50 C for 1 min,
15 C/min to 180 C, hold 0 min
7 C/min to 230, hold 0 min
30 C/min to 380, hold 10 min

3
Results and Discussion Using the approach detailed in the ASTM and CEN
methods, the amount of glycerin in each sample
After running the standards, Agilent ChemStation was calculated with the calibration functions
was used to calculate linear calibration curves for derived from the glycerin calibration curve. Like-
glycerin, monoolein, diolein, and triolein. The wise, the amount of monoglycerides, diglycerides,
curves for each compound showed excellent linear- and triglycerides was determined from the
ity and y-intercepts near zero. These curves are monoolein, diolein, and triolein calibration func-
shown in Figure 1. The correlation coefficients (r2) tions, respectively. Table 6 list the amounts of glyc-
for each compound exceeded the specification of erin and glycerides found in each sample.
0.99 set forth in the ASTM and CEN methods.
Precision of the analysis was measured using
Figure 2 shows the typical chromatograms repeatability, which is the difference between two
obtained for samples of soybean B100 and rape- successive analyses of the same sample run on the
seed B100. The large peaks observed in each chro- same day by a single operator on the same instru-
matogram are the FAMEs present in the samples. ment. This repeatability measurement was made
Figure 3 shows the selected regions of the rapeseed for each sample over four consecutive days. Table 7
chromatogram where glycerin, monoglycerides, shows the results of the daily precision measure-
diglycerides, and triglycerides elute. Peak identifi- ments compared to the specifications from the
cation for each compound is made using the rela- ASTM D6584 method. These results show excellent
tive retention times published in the ASTM method single-day precision as determined by repeatabil-
(Table 5). The retention time of the first internal ity.
standard, 1,2,4-butanetriol, was used to identify
glycerin. The retention time of the second internal ASTM D6584 and EN14105 are not easy methods
standard, tricaprin, was used to identify the mono- to run for a number of reasons: the sample prepa-
glycerides, diglycerides, and triglycerides. ration is lengthy and difficult; the sample injection

Area ratio 5 Area ratio 5

0.5 Glycerin calibration Monoolein calibration
1.5 4
0.4 4
1.25
0.3 3 1.0 3

0.2 2 0.75
2
0.5
0.1 1 1
Y = 1.0351* X + 0.0010 0.25 Y = 1.3458* X + 0.0039
0 r2: 0.9999 0 r2: 0.9997
0 0.2 Amount ratio 0 0.5 1 Amount ratio

Area ratio 5 Area ratio

Diolein calibration Triolein calibration 5
0.4
0.7
0.6 4 4
0.3
0.5
0.4 3 0.2 3
0.3
0.2 2 0.1 2
0.1 1 Y = 1.18629* X - 0.0080 1 Y = 0.6846* X - 0.0059
0 r2: 0.9999 0 r2: 0.9998
0 0.2 0.4 Amount ratio 0 0.2 0.4 Amount ratio

Figure 1. Calibration curves for glycerin, monoolein, diolein, and triolein.

4
 Soybean Biodiesel
pA

Tricaprin (istd 2)
1000

600 Butanetriol (istd 1)

200

2.5 5 7.5 10 12.5 15 17.5 20 min

Rapeseed Biodiesel

pA
Tricaprin (istd 2)
1000

600 Butanetriol (istd 1)

200

2.5 5 7.5 10 12.5 15 17.5 20 min

Figure 2. Chromatograms showing typical analysis of free and total glycerins in two B100 biodiesel samples.

200 300
Monoolein, monolinolein,
Butanetriol (istd 1) and monolinolenin
150
200

100
Monopalmitin Monostearin
Glycerin 100
50

0 0
4 4.5 5 5.5 6 6.5 min 14 14.5 15 15.5 16 16.5 min

250 Diglycerides
120
200

150 80 Triglycerides
100
40
50

0 0
20 20.2 20.4 20.6 20.8 min 21 21.5 22 22.5 min

Figure 3. Details of glycerin, monoglycerides, diglycerides, and triglycerides found in a sample of rapeseed B100 biodiesel.

5
onto a 0.32-mm id column is not easily automated; since high methanol content or water content
and calibration can be difficult. However, there are will inhibit derivatization.
a number of guidelines and procedures that can be
6. Run all samples immediately after preparation.
followed to obtain good, precise results.
Do not store prepared sample for more than
several hours, especially in humid environ-
Sample and Standard Preparation
ments.
1. Prepare fresh calibration standards every day.
Once the standards are prepared they should GC Analysis
not be stored for more than several hours.
It is recommended that a retention gap be used
2. Use commercially prepared stock or final cali- between the GC inlet and the column. The reten-
bration standards packaged in sealed, glass tion gap will improve peak shape and sample
ampoules. If all of the standard solutions are vaporization, as well as maintain column effi-
not used in a single day, do not save for later ciency. Figure 4 shows the improvement in peak
use. Water can accumulate in the solutions and shape for glycerin and 1,2,3-butanetriol when
this will inhibit derivatization. using a 0.53-mm id retention gap. A retention gap
3. Only use derivatization-grade MSTFA. Lesser will also prolong the column life since it traps any
grades contain solvents that can reduce the nonvolatile compound contained in the sample. A
effectiveness of the reagent. It is best to pur- 0.53-mm id retention gap will also make sample
chase MSTFA in small quantities packaged in injection easier since it can easily accommodate
sealed, glass ampoules. As with the standards, the standard single tapered syringe needle.
discard any unused MSTFA.
4. Use only clean, dry glassware and pipettes. Table 6. Weight Percent of Free and Total Glycerin

5. Only analyze finished product B100. This %(m/m) in Soybean B100 Biodiesel
method should not be used for process samples Day 1 Day 2 Day 3 Day 4
(avg)* (avg)* (avg)* (avg)*
Table 5. Relative Retention Times Used for Peak Identification Free glycerin 0.004 0.004 0.004 0.004
Monoglycerides 0.287 0.280 0.285 0.290
RRT RRT Diglycerides 0.533 0.527 0.533 0.546
(int std 1) (int std 2) Triglycerides 0.387 0.371 0.340 0.304
Glycerin 0.85
1,2,3-Butanetriol (int std 1) 1.00 %(m/m) in Rapeseed B100 Biodiesel
Monopalmitin 0.76 Day 1 Day 2 Day 3 Day 4
Monoolein, monolinolein, 0.83 0.86 (avg)* (avg)* (avg)* (avg)*
monolinolenin, monostearin Free glycerin 0.002 0.002 0.002 0.002
Tricaprin (int std 2) 1.00 Monoglycerides 0.365 0.375 0.370 0.371
Diglycerides 1.05 1.09 Diglycerides 0.256 0.262 0.256 0.256
Triglycerides 1.16 1.31 Triglycerides 0.021 0.019 0.018 0.016
*Average of 2 runs per day for each sample.

Table 7. Repeatability Results for Two B100 Biodiesel Samples Over Four Days
Soybean B100 Biodiesel
ASTM D6584
Specification Observed repeatability (%m/m)
(% m/m) Day 1 Day 2 Day 3 Day 4
Glycerin 0.001 0.000 0.000 0.000 0.000
Monoglycerides 0.021 0.005 0.007 0.007 0.000
Diglycerides 0.021 0.008 0.008 0.014 0.000
Triglycerides 0.032 0.008 0.004 0.005 0.000

Rapeseed B100 Biodiesel

ASTM D6584
Specification Observed repeatability (%m/m)
(% m/m) Day 1 Day 2 Day 3 Day 4
Glycerin 0.001 0.000 0.000 0.000 0.000
Monoglycerides 0.021 0.007 0.000 0.006 0.000
Diglycerides 0.021 0.003 0.002 0.000 0.000
Triglycerides 0.032 0.002 0.000 0.001 0.000
6
One problem with using a retention gap is the high
oven temperature (380 C) required for triglyc-
eride elution. Most fused-silica tubing cannot be
used above 350 C. Also, traditional column
unions can leak above that temperature. The Agi-
lent Capillary Flow Technology Ultimate Union
combined with special high-temperature fused-
silica tubing can solve this problem. The Ultimate
Union is made with deactivated stainless steel that
can be taken to 400 C without losing inertness. DB-5ht Column bracket
The high-temperature polyimide coating on the
retention gap has extended lifetime up to 380 C.

Successfully using this Union first requires that

the retention gap and column be correctly
installed using the metal ferrules designed for the
Union. Next, the Union must be completely sup-
ported so that no weight is placed on the column
connections. A bracket is supplied with the Ulti- Ultimate Union
mate Union Kit to support the union fitting to the
GC oven wall. Failure to do this will result in a
large leak after only a few runs above 350 C,
resulting in column damage. Figure 5 shows a cor-
rect installation with the Union supported on its
bracket in the GC oven. From this photo it can be
seen there is no stress on the column or retention Retention gap
gap. Additionally, to extend the lifetime of this
connection, the oven temperature should be kept
at 50 C between analyses. It is also recommended
that the Union be checked for leaks before running Figure 5. Details of the retention gap and analytical column
joined with a Capillary Flow Technology Ultimate
Union.
Butanetriol (istd 1)

samples. If a leak is detected, make a new connec-

tion to the Union with a new ferrule, and evaluate
the column performance before running samples.

No Retention Gap Conclusions

The analysis of free and total glycerins can be done
using ASTM D6584 or EN14105. Both methods are
nearly identical in sample preparation and analy-
Glycerin
sis. This application described the configuration of
an Agilent 7890A gas chromatograph for these
methods. By combining careful and deliberate
sample preparation with a high-temperature reten-
tion gap and a Capillary Flow Technology Ultimate
Union, this system can obtain results that meet or
1 m x 0.53 mm I.D.
exceed the methods calibration and precision
Retention Gap
specifications.

Figure 4. Improved peak shape for glycerin and 1,2,3-

butanetriol when using a retention gap and the Cap-
illary Flow Technology Ultimate Union.

7
 www.agilent.com/chem
References 4. D6585 Test Method for Determination of Free
and Total Glycerine in B-100 Biodiesel Methyl
1. K. Shaine Tyson, Biodiesel Handling and Use Esters by Gas Chromatography, ASTM Interna-
Guidelines, National Renewable Energy Labo- tional, 100 Bar Harbor Drive, West Con-
ratory, NREL/TP-580-30004, September 2001 shohocken, PA, USA, 2003
2. D6751 Standard Specification for Biodiesel 5. EN14105 Fat and Oil DerivativesFatty Acid
Fuel Blend Stock (B100) for Middle Distillate Methyl Esters (FAME)Determination of Free
Fuel, ASTM International, 100 Bar Harbor and Total Glycerol and Mono-, Di- and Triglyc-
Drive, West Conshohocken, PA 19428 USA eride Content, European Committee for Stan-
3. EN14214 Fatty Acid Methyl Esters (FAME) for dardization: Management Centre, rue de
Diesel Engines, Requirements and Test Meth- Stassart 36, B-1050 Brussels, 2003
ods, European Committee for Standardization:
Management Centre, rue de Stassart 36, B-1050 For More Information
Brussels, 2003
For more information on our products and services,
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Information, descriptions, and specifications in this publication are subject to change

without notice.

Agilent Technologies, Inc. 2007

Printed in the USA

November 9, 2007
5989-7269EN