AppNote 1/2010
Full Automation of ASTM Method
D6584-07 “Standard Test Method for
the Determination of Free and Total
Glycerin in B-100 Biodiesel Methyl
Esters by Gas Chromatography” using
a GERSTEL Dual Rail PrepStation
John R. Stuff, Jacqueline A. Whitecavage
Gerstel, Inc., 701 Digital Dr. Suite J,
Linthicum, MD 21090, USA
KEYWORDS
Biodiesel, Automation, ASTM D6584-07
ABSTRACT
Biodiesel is defi ned as mono-alkyl esters of long chain
fatty acids derived from vegetable oils or animal fats
which conform to ASTM D6751 specifi cations for use in
diesel engines. Biodiesel is produced from the fat or oil by
transesterifi cation. Glycerin is an unwanted byproduct of the
reaction and must be removed from the fi nal product.
ASTM Method D6584-07 measures the amount of residual
free and bonded (mono-, di-, and triglycerides) glycerin in
the biodiesel fuel. Standard and sample preparation, which
requires a derivatization step, can be laborious and time
consuming.
Using the GERSTEL MPS 2 Dual Rail PrepStation and
MAESTRO control software, the entire method, including
standards and sample preparation, derivatization, dilution,
and analysis can be fully automated. This paper shows the
details of the automation.
AN/2010/1 - 2
INTRODUCTION
A dual rail PrepStation was set up to fully automate
the procedure outlined in ASTM D6584-07 for the
determination of free and total glycerides in B-
100 biodiesel fuel. The goal is to provide complete
automation of standard preparation, automate next
sample preparation within the time frame of the GC
run, and provide other user-friendly features of the
Maestro software control for the ASTM method.
Full automation frees up the analyst’s time for other
laboratory operations.
EXPERIMENTAL
Instrumentation. Analyses were performed on an
Agilent Technologies 6890 GC with fl ame ionization
detector (FID), PTV inlet (CIS 4, GERSTEL) and Dual
Rail MPS 2 robotic sampler with 10 μL on-column
syringe and an 80 μL sideport syringe with diluter
module (GERSTEL).
Analysis conditions.
PTV: On-Column
60°C (0.05 min); 0.2°C/sec;
230°C (2 min); 0.5°C/sec;
380°C (10 min)
Column:
10 m Rtx-Biodiesel TG (Restek)
di = 0.32 mm df = 0.1 μm
with 2m integrated guard column,
di = 0.53 mm
Pneumatics: He, constant fl ow = 3 mL/min
Oven:
50°C (1 min); 15°C/min;
180°C;
7°C/min;
230°C; 30°C/min; 380°C (10 min)
FID: 380°C
Standard description. A biodiesel standards kit
(#D6584-SS), containing separate 5 mL ampoules of
500 ng/μL glycerin, 5000 ng/μL monoolein, 500 ng/μL
diolein, 5000 ng/μL triolein, 1000 ng/μL butanetriol,
and 8000 ng/μL tricaprin, all in pyridine, was purchased
from Analytical Services, Inc. (The Woodlands, TX).
The kit also contained MSTFA and heptane. Biodiesel
B-100 was purchased locally.
Sample preparation. The glycerin, mono-, di- and
triolein standards were placed in 2 mL screw cap
vials in Positions 1-4, respectively, of a VT-98 tray.
The butanetriol, tricaprin and MSTFA were put in 10
mL vials with PTFE snap caps and placed in Positions
3, 4, and 5 in the 5 position 10 mL wash tray. Empty
10 mL screw cap vials were placed in Positions 1-
5 of the VT-32-10 tray. Biodiesel B-100 samples,
approximately 100 mg, were weighed directly into
10 mL screw cap vials and placed on the VT-32-10
tray. The samples and standards were prepared using
a GERSTEL MAESTRO PrepSequence.
Sample introduction. The samples were prepared in
vials placed in the VT32-10 tray. One microliter of the
standard or sample was injected into the CIS 4 using the
MPS 2 autosampler with a 10 μL liquid syringe with
replaceable 26 gauge needle for on-column injection
RESULTS AND DISCUSSION
The GC system used for this study is shown in Figure
1. The method calls for the preparation of a fi ve point
calibration curve for glycerin, mono-, di-, and triolein
from the stock standards. The required volumes are
shown in Table 1.
Figure 1. System setup.
Standard Number
1
2
3
4
5
µL Glycerin Stock
10
30
50
70
100
µL Monoolein Stock
20
50
100
150
200
µL Diolein Stock
10
20
40
70
100
µL Triolein Stock
10
20
40
70
100
Table 1. Required stock standard volumes.
AN/2010/1 - 3
The GERSTEL MAESTRO PrepBuilder function is used to automate the preparation and injection of standards
and samples. An example of a standard preparation loop for the addition of the diolein stock to the fi ve standard
vials is shown below. The individual steps are selected by mouse-click from a drop-down menue.
The fi rst 2 ADD steps correspond to the addition of
100 μL of the butanetriol internal standard. After the
syringe is washed, 100 μL of tricaprin internal standard
is added, followed by another wash and 100 μL of the
MSTFA to derivatize the sample/standard. The sample
or standard is then mixed, a 15 minute wait step is
inserted to allow the derivatization to be completed,
heptane is added for dilution, followed by mixing and
GC analysis.
ACTION
METHOD / VALUE
SOURCE
DESTINATION
PREP Vials 3-3
Extensive
(R) ADD
Add 10 uL Std
3 @ Tray2,VT98
1 @ Tray1,VT32-10
(R) ADD
Add 20 uL Std
3 @ Tray2,VT98
2 @ Tray1,VT32-10
(R) ADD
Add 40 uL Std
3 @ Tray2,VT98
3 @ Tray1,VT32-10
(R) ADD
Add 70 uL Std
3 @ Tray2,VT98
4 @ Tray1,VT32-10
(R) ADD
Add 50 uL Std
3 @ Tray2,VT98
5 @ Tray1,VT32-10
(R) ADD
Add 50 uL Std
3 @ Tray2,VT98
5 @ Tray1,VT32-10
(R) DILUTE
Dilute Wash Syringe
Waste
END
ACTION
METHOD / VALUE
SOURCE
DESTINATION
PREP Vials 1-10
Extensive
(R) ADD
ADD First 50 uL
SolvRes1
Tray1,VT32-10
(R) ADD
ADD Second 50 uL
SolvRes1
Tray1,VT32-10
(R) DILUTE
Dilute Wash Syringe
Waste
(R) ADD
ADD First 50 uL
SolvRes2
Tray1,VT32-10
(R) ADD
ADD Second 50 uL
SolvRes2
Tray1,VT32-10
(R) DILUTE
Dilute Wash Syringe
Waste
(R) ADD
ADD First 50 uL
SolvRes3
Tray1,VT32-10
(R) ADD
ADD Second 50 uL
SolvRes3
Tray1,VT32-10
(R) DILUTE
Dilute Wash Syringe
Waste
(R) MOVE
Tray1,VT32-10
Agitator,AgiTray
(R) MIX
Mix 1 Minute
(R) MOVE
Agitator,AgiTray
Tray1,VT32-10
(R) WAIT
15.00 min
(R) DILUTE
Dilute 8 mL Heptane
Tray1,VT32-10
(R) MOVE
Tray1,VT32-10
Agitator,AgiTray
(R) MIX
Mix 1 Minute
(R) MOVE
Agitator,AgiTray
Tray1,VT32-10
(L) INJECT
ASTM_D6584.m\maestro.mth
Tray1,VT32-10
Rear
END
Samples are prepared by weighing approximately 100 mg into an 10 mL empty vial. The preparation of standards
and samples is performed as follows:
1. Add
100
μL each of 2 Internal Standards
2. Add
100
μL Derivatizing Reagent, Mix, Wait 15 min
3.
Dilute with 8 mL heptane, Mix
4.
Analyze using a 1 μL on-column Injection
An example for the preparation and analysis loop of the fi ve standards and fi ve samples is shown below.
The inject action step contains all the Agilent
Chemstation and GERSTEL MPS 2 functions required
for analysis of the samples. When integrated into
Agilent ChemStation or MS ChemStation, one method
and one sequence table runs the complete system.
AN/2010/1 - 4
Table 2. Regression data for standards.
Figure 3. Diolein calibration curve.
Figure 2 shows a typical chromatogram obtained for Standard #3. The standard and internal standard peaks
are labeled in the chromatogram. Table 2 shows the linear regression results for the standard curves. Good
correlation is achieved for all standards. Figure 3 shows the calibration curve for diolein.
Figure 2. Chromatogram for standard #3.
pA
100
400
Time-->
10.00
15.00
Glycer
in
5.00
20.00
200
300
500
25.00
700
600
Butanetr
iol
Monoolein
Tr
icapr
in
Diolein
Tr
iolein
0 4
0.5
0.6
0.7
0
0.1
0.2
0.3
.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Compound
r2
m
b
Glycerin
0.9999
1.181
-0.0008
Monoolein
0.9997
1.247
-0.0122
Diolein
0.9987
1.0203
-0.0067
Triolein
0.9985
0.8946
-0.0120
AN/2010/1- 5
Figure 4. Chromatogram for biodiesel sample.
Table 3 shows the results for the sample replicates (n=5). Reproducibilities are good with %RSDs ranging from
2.1-3.4%. A typical chromatogram for this sample type is shown in Figure 4.
Table 3. Mass percentages for B-100 Biodiesel.
Glycerin
Monoglycerides
Diglycerides
Triglycerides
Sample 1
0.02608
1.107
0.1898
0.08909
Sample 2
0.02530
1.055
0.1864
0.09379
Sample 3
0.02662
1.059
0.1890
0.09398
Sample 4
0.02706
1.056
0.1858
0.09565
Sample 5
0.02670
1.071
0.1791
0.08861
Average
0.02635
1.070
0.1860
0.09220
Std Dev
0.000683
0.0220
0.00421
0.00316
%RSD
2.59
2.06
2.26
3.42
pA
100
400
Time-->
10.00
15.00
Glycer
in
5.00
20.00
200
300
500
25.00
700
600
Butanetr
iol
Monoglycer
ides
Tr
icapr
in
Diglycer
ides
Tr
iglycer
ides
AN/2010/1 - 6
Figure 5. MAESTRO sequence scheduler
CONCLUSIONS
A dual rail PrepStation was programmed to prepare standards and samples, and inject samples using GERSTEL
MAESTRO software. The results showed good linearity for the standards and good reproducibility (%RSDs from
2.1-3.4%, n=5) for the B-100 biodiesel samples. Complete automation of ASTM D6584-07 was accomplished
with next sample preparation within the timeframe of the GC run.
The preparation of each sample was completed in approximately 27 minutes using MAESTRO PrepBuilder
functions, well within the total GC cycle time of 38 minutes (31 minute GC run + 7 minute cooldown). This
means that samples can be prepared during the GC run of the preceding sample without adding to the overall
analysis time, a fact which is clearly illustrated by the MAESTRO Sequence Scheduler window (Figure 5).
The scheduler graphically illustrates that the sample prep and GC run segments of the analysis are performed
in parallel, while giving a clear overview of the total analysis time needed in order to facilitate laboratory
workfl ow planning.
AN/2010/1 - 7
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