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Designation: D6584 17
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D6584 17
TABLE 3 Standard Solutions TABLE 4 Approximate Relative Retention Times
Standard Solution Number 1 2 3 4 5 Component Use Internal Relative Retention
L of glycerin stock solution 10 30 50 70 100 Standard Time
L of monoolein stock solution 20 50 100 150 200 Glycerin 1 0.85
L of diolein stock solution 10 20 40 70 100 1,2,4 Butanetriol 1.00
L of triolein stock solution 10 20 40 70 100 Internal Standard 1
L of butanetriol stock solution 100 100 100 100 100 Monopalmitin 2 0.76
L of tricaprin stock solution 100 100 100 100 100 Monoolein, monolinolein 2 0.83-0.86
monolinolenin, and monostearin
Tricaprin 1.00
Internal Standard 2
where: Diglycerides 2 1.05-1.09
Triglycerides 2 1.16-1.31
Ai = area of reference substance, and
As = area of internal standard.
amti 5 ~ W i /W s ! (2) 10.2 Inject 1 L of the reaction mixture into the cool
where: on-column injection port and start the analysis. Obtain a
chromatogram and peak integration report.
Wi = mass of reference substance, and
Ws = mass of internal standard. 10.3 Peak IdentificationIdentify peaks by comparison of
retention times to the standards. For identification of additional
9.4.1 Prepare a calibration curve for each reference compo-
peaks, use the relative retention times given in Table 4 and the
nent by plotting the response ratios (rspi), as the y-axis, versus
reference chromatograms given in Fig. 1. Mono-, di-, and
the amount ratios (amti), as the x-axis.
triglycerides are separated according to carbon numbers (CN).
9.5 Calculate the correlation coefficient r2 value for each
10.4 Monoglyceride consists of the four overlapping peaks
reference component in the calibration set using Eq 3. The r2
with relative retention times (RRT) of 0.76 and 0.83 to 0.86
value should be at least 0.99 or greater. If the above criteria for
with respect to the internal standard tricaprin. A pair of peaks,
r2 are not met, rerun the calibration or check instrument
methyl esters with a carbon number of 24, may appear with
parameters and hardware.
RRT of 0.80 to 0.82, and should not be included in the
~ ( xy! 2 calculation of monoglyceride.
r2 5 (3)
~ ( x 2 !~ ( y 2 ! 10.5 Diglyceride is also primarily separated according to
where: carbon number, but due to varying double bonds in the
molecules, baseline resolution of the peaks does not occur. The
x 5 X i 2 x (4)
grouping of 3 to 4 peaks with RRT of 1.05 to 1.09 (CN 34, 36,
y 5 Y i 2 y (5) and 38) shall be attributed to diglyceride. Carbon number also
and: separates triglyceride. Peaks with RRT of 1.16 to 1.31 (CN 52,
54, 56, and 58) should be included in the calculation.
Xi = amti ratio data point,
x = average values for all amti data points 11. Calculation and Report
Yi = corresponding rspi data points,
y = average values for all rspi data points. 11.1 After identifying the peaks, measure the areas of the
peaks identified as glycerin, mono-, di-, and triglyceride. Using
9.6 Calibration FunctionsFor each reference calibration the slope and y-intercept of the calibration functions, calculate
functions are calculated in the form: the mass of each as follows:
Ax
A is
5 axF S DG
Wx
W is
1b x (6)
11.1.1 Glycerin:
where:
G5 F GSF G DF G
W is1
ag
Ag
A is
2 bg
100
W
(7)
standard and MSTFA. Shake the vials, and allow to set for where:
15 min to 20 min at room temperature. Add approximately
8 mL of n-Heptane to the vial and shake. Glj = mass percentage of individual glyceride in sample,
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D6584 17
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D6584 17
r 5 0.195E 201 ~ FG10.0001! 0.27 (11) R = reproducibility.
where: 12.1.2.2 Free Glycerin Reproducibility:
FG = the calculated result for free glycerin expressed as a
R 5 0.246E 201 ~ FG10.0001! 0.27 (16)
mass % of the glycerin content in the sample, and
r = repeatability. where:
12.1.1.3 Total Monoglyceride Repeatability: FG = the calculated result for free glycerin expressed as a
r 5 0.78E 201M 0.62 (12)
mass % of the glycerin content in the sample, and
R = reproducibility.
where:
12.1.2.3 Total Monoglyceride Reproducibility:
M = total monoglyceride concentration in mass %, and
r = repeatability. R 5 0.46M 0.62 (17)
12.1.1.4 Total Diglyceride Repeatability: where:
r 5 0.344D 0.93 (13) M = total monoglyceride concentration in mass %, and
R = reproducibility.
where:
D = total diglyceride concentration in mass %, and 12.1.2.4 Total Diglyceride Reproducibility:
r = repeatability. R 5 0.784D 0.93 (18)
12.1.1.5 Total Triglyceride Repeatability: where:
r 5 0.12T 0.687 (14) D = total diglyceride concentration in mass %, and
where: R = reproducibility.
T = total triglyceride concentration in mass %, and 12.1.2.5 Total Triglyceride Reproducibility:
r = repeatability.
R 5 1.08T 0.687 (19)
12.1.2 ReproducibilityThe difference between two single
and independent results, obtained by different operators work- where:
ing in different laboratories on identical material, would in the T = total triglyceride concentration in mass %, and
long run, in the normal and correct operation of the test R = reproducibility.
method, exceed the following values only in one case in
12.2 BiasSince there is no accepted reference material
twenty.
suitable for determining bias for the procedure in this test
12.1.2.1 Total Glycerin Reproducibility:
method, bias can not be determined.
R 5 0.29TG 0.73 (15)
APPENDIX
(Nonmandatory Information)
X1. REFERENCE MIX AND EXAMPLE CHROMATOGRAMS FOR IMPROVED PEAK IDENTIFICATION
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D6584 17
FIG. X1.1 Overlay of Canola based B100 and Monoglyceride Standard Solution
chromatograms, except where noted, were collected using determine the exact retention times of the monoglyceride
helium carrier gas setting of constant flow of 3 mL min. This species, which can then be applied to the B100 chromatogram
is done to aid in consistency of the example chromatograms. to assist in peak identification. Figs. X1.1 and X1.2 show an
Chromatograms may differ in different laboratories from those overlay of a commercially prepared monoglyceride standard
shown, depending on the chromatographic system and configu- and two typical B100 chromatograms.
ration used. X1.3.1.1 Monoglyceride MixtureA standard monoglycer-
ide mix, containing monoolein, monostearin, and monopalmi-
X1.2 Reagents and Materials tin should be prepared. The mixture may be purchased com-
X1.2.1 Purity of ReagentsReagent grade chemicals shall mercially or prepared prior to analysis. In either case, the
be used in all tests. Unless otherwise indicated, it is intended monoglycerides should be roughly 100 mg each dissolved in
that all reagents conform to the specifications of the Committee 10 mL pyridine. This mixture will be used to make the
on Analytical Reagents of the American Chemical Society standard.
where such specifications are available.3 Other grades may be X1.3.2 Preparation of Monoglyceride StandardTransfer
used provided it is first ascertained that the reagent is of 100 L of monoglyceride mixture (X1.3.1.1) and 100 L
sufficient purity to permit its use without lessening the accu- MSTFA into a 10 mL screw cap vial. Avoid contact with
racy of the determination. moisture. Close vial with PTFE-lined lid and shake. Allow the
X1.2.2 1-Monooleoyl-rac-glycerol (monoolein), CAS 111- vial to stand for 15 min to 20 min at room temperature. Add
03-5. approximately 8 mL n-heptane to the vial and shake.
X1.2.3 1-Monopalmitoyl-rac-glycerol (monopalmitin), re- X1.3.3 Chromatographic AnalysisIf using an automatic
agent grade, CAS 542-44-9. sampler, transfer an aliquot of the solution into a glass GC vial
and seal with a PTFE-fluorocarbon lined cap. Analyze the
X1.2.4 1-Monostearoyl-rac-glycerol (monostearin), CAS monoglyceride standard following Section 10, Procedure.
123-94-4.
X1.2.5 Pyridine, reagent grade. X1.4 Variations in Chromatography Due to Constant
Pressure Operation Compared to Constant Flow
X1.2.6 N-Methyl-N-trimethylsilultrifluoroacetamide Operation
(MSTFA), reagent grade.
X1.4.1 Options in many gas chromatographs may allow for
X1.2.7 n-Heptane, reagent grade. the carrier gas flow to be defined by constant pressure or
constant flow. Electronic pressure control, found on many
X1.3 Preparation of Monoglyceride Standard newer systems, may allow for constant flow or constant
X1.3.1 The use of a reference standard of monoglycerides is pressure operation. In general, older systems, unless modified
strongly suggested for identification of the individual mono- to include electronic pressure control, may only allow for
glycerides in B100. This reference standard can be used to constant pressure operation.
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D6584 17
FIG. X1.2 Overlay of Soy based B100 and Monoglyceride Standard Solution
X1.4.2 Depending on the operation of the chromatographic X1.5 Additional Example Chromatograms
system, the regions of interest (monoglyceride, diglyceride,
X1.5.1 All samples were run using helium carrier gas with
and triglyceride) may not match those outlined in the method.
Fig. X1.3 illustrates a sample injected, first using constant flow constant flow through the column of 3 mL min. Figs. X1.4-
control, then using constant pressure control; all other condi- X1.6 provide example chromatograms of various B100 feed-
tions were identical between runs. Note that the retention times stocks to show some of the variability that may be experienced
of the peaks shift depending on the operation. when running this method. Peak identifications are also shown.
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D6584 17
FIG. X1.3 Overlay of B100 sample derived from mixed feedstocks. The top chromatogram was collected with constant flow setting at
3 mL min and bottom chromatogram was collected with constant pressure, measured as 3 mL min at 50 C. All other parameters re-
mained the same
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D6584 17
SUMMARY OF CHANGES
Subcommittee D02.04 has identified the location of selected changes to this standard since the last issue
(D6584131) that may impact the use of this standard. (Approved June 1, 2017.)
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