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 defined as mono-alkyl esters of long chain
fatty acids derived from vegetable oils or animal fats
which conform to ASTM D6751 specifications for use in
diesel engines. Biodiesel is produced from the fat or oil by
transesterification. Glycerin is an unwanted byproduct of the
reaction and must be removed from the final 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.
INTRODUCTION 5 of the VT-32-10 tray. Biodiesel B-100 samples,
A dual rail PrepStation was set up to fully automate approximately 100 mg, were weighed directly into
the procedure outlined in ASTM D6584-07 for the 10 mL screw cap vials and placed on the VT-32-10
determination of free and total glycerides in B- tray. The samples and standards were prepared using
100 biodiesel fuel. The goal is to provide complete a GERSTEL MAESTRO PrepSequence.
automation of standard preparation, automate next
sample preparation within the time frame of the GC Sample introduction. The samples were prepared in
run, and provide other user-friendly features of the vials placed in the VT32-10 tray. One microliter of the
Maestro software control for the ASTM method. standard or sample was injected into the CIS 4 using the
Full automation frees up the analyst’s time for other MPS 2 autosampler with a 10 μL liquid syringe with
laboratory operations. replaceable 26 gauge needle for on-column injection

EXPERIMENTAL RESULTS AND DISCUSSION

Instrumentation. Analyses were performed on an The GC system used for this study is shown in Figure
Agilent Technologies 6890 GC with flame ionization 1. The method calls for the preparation of a five point
detector (FID), PTV inlet (CIS 4, GERSTEL) and Dual calibration curve for glycerin, mono-, di-, and triolein
Rail MPS 2 robotic sampler with 10 μL on-column from the stock standards. The required volumes are
syringe and an 80 μL sideport syringe with diluter shown in Table 1.
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 flow = 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 Figure 1. System setup.
diolein, 5000 ng/μL triolein, 1000 ng/μL butanetriol,
and 8000 ng/μL tricaprin, all in pyridine, was purchased Table 1. Required stock standard volumes.
from Analytical Services, Inc. (The Woodlands, TX).
The kit also contained MSTFA and heptane. Biodiesel Standard Number 1 2 3 4 5
B-100 was purchased locally. µL Glycerin Stock 10 30 50 70 100
Sample preparation. The glycerin, mono-, di- and µL Monoolein Stock 20 50 100 150 200
triolein standards were placed in 2 mL screw cap µL Diolein Stock 10 20 40 70 100
vials in Positions 1-4, respectively, of a VT-98 tray. µL Triolein Stock 10 20 40 70 100
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-

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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 five standard
vials is shown below. The individual steps are selected by mouse-click from a drop-down menue.
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

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 five standards and five samples is shown below.

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

The first 2 ADD steps correspond to the addition of The inject action step contains all the Agilent
100 μL of the butanetriol internal standard. After the Chemstation and GERSTEL MPS 2 functions required
syringe is washed, 100 μL of tricaprin internal standard for analysis of the samples. When integrated into
is added, followed by another wash and 100 μL of the Agilent ChemStation or MS ChemStation, one method
MSTFA to derivatize the sample/standard. The sample and one sequence table runs the complete system.
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.
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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.
pA

700

Tricaprin
600

500

400

Monoolein
300
Butanetriol

Diolein

Triolein
200
Glycerin

100

Time--> 5.00 10.00 15.00 20.00 25.00

Figure 2. Chromatogram for standard #3.

Table 2. Regression data for standards.

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

0.7

0.6

0.5

04
0.4

0.3

0.2

0.1

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Figure 3. Diolein calibration curve.

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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

700

Tricaprin
600

500
Monoglycerides

400

300
Diglycerides
Butanetriol

Triglycerides

200
Glycerin

100

Time--> 5.00 10.00 15.00 20.00 25.00

Figure 4. Chromatogram for biodiesel sample.

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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
workflow planning.

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.

AN/2010/1 - 6
AN/2010/1 - 7
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