5
Table 3. A final “script” showing each step in the sample prep protocol and the corresponding WorkBench methods needed to perform each action.
C. Multiuser precision - reproducibility In order to evaluate multi-user precision, four different chemists were provided with a soybean biodiesel sample, calibration standards and a WorkBench with the chemical resources shown in Table 2. Each chemist was given the list of sample preparation steps outlined in Table 3 and asked to develop and use a WorkBench protocol. Duplicates of a soybean biodiesel sample were prepared using their WorkBench followed by GC analysis.
Results A. Preparation of GC calibration standards
The 5-level calibration curves for glycerin, monoolein, diolein and triolein are shown in Figure 3. The five standards used to create these curves were prepared with the Agilent WorkBench. The glycerin curve was used to quantify free glycerin in the biodiesel samples. The monoolein curve was used for the monoglycerides, the diolein curve for all diglycerides and the triolein curves for all triglycerides found in the samples. The same calibration standards were also prepared manually and used to construct calibration curves. In Table 6 we compared the calibration models for all four compounds from the manually prepared standards and the WorkBench prepared standards. The manually prepared standards and the WorkBench prepared standards yielded nearly identical calibration curves and the correlation coefficients (r2
) from the WorkBench prepared standards exceeded the ASTM specification of at least 0.99 or greater.
Figure 2. The WorkBench Sequence Queue is used to run the WorkBench methods described in Table 3.
Experimental
An Agilent 7890A GC was configured to run ASTM D6584. This configuration is outlined in Table 4. The GC conditions used to analyse the biodiesel samples and standards are shown in Table 5.
Table 4. Gas chromatographic instrument configuration used to analysed samples using ASTM method.
Standard 7890A GC Hardware G3440A
Option 122 Option 211 G4513A
Columns Analytical Column Select Biodiesel for Glycerides 15m x 0.32mm ID x 0.1 um film (part no. cp9078) Data System Agilent Multi-Technique Chemstation
Consumables 5181-1267
10 uL Teflon fixed autoinjector syringe
Standards and Reagents 5190-1408 5190-1407
Biodiesel D6584 Calibration Standards Kit
Biodiesel MSTFA Kit Reagent grade n-heptane
Table 5. GC instrument conditions for ASTM method D6584.
Cool-on-column inlet Initial temperature Temperature program
Column flow
Column Temperature Initial Rate 1 Rate 2 Rate 3
50 deg C Oven track mode
Helium at 3 mL/min. constant flow mode 50 deg C for 1 min.
15 deg C/min. to 180 deg C, hold 0 min. 7 deg C/min. to 230 deg C, hold 0 min. 30 deg C/min. to 380 deg C, hold 10 min.
Flame ionisation detector 380 deg C pA A. Preparation of GC calibration standards
ASTM D6584 also specifies the derivatisation of five calibration standards with the same preparation used for the samples. After running the standards by GC, the resulting calibration curves were evaluated for linearity before running any samples. The calibration standards were prepared both manually and with the WorkBench with the same protocol used for the samples. The calibration curves resulting from the manual prep were used to quantify the manually prepared biodiesel samples. The calibrations resulting from the WorkBench prepared standards were used to quantify the WorkBench prepared samples.
B. Comparison of manual sample prep and WorkBench sample prep The first question many users will ask is ‘does a scaled WorkBench sample prep produce the same results as the manual sample prep’. To help answer that question, two different types of biodiesel samples were prepared using the manual ASTM protocol and the WorkBench. The first biodiesel sample came from a small local producer using canola oil as the feedstock. The second sample was supplied by a national producer using a soybean oil feedstock. For both the manual and WorkBench protocols, each biodiesel sample was prepared and analysed in duplicate to evaluate the repeatability (single user precision) according to the ASTM method.
200 400 600 800 1000
0
200 400 600 800 1000
0 0 5 10 15 20 25 Min.
Figure 4. A comparison of data from a canola biodiesel sample prepared manually and using the Agilent WorkBench. These chromatograms show remarkable similarity in the four regions where glycerin, monoglycerides, diglycerides and triglycerides are separated.
glycerin monoglycerides diglycerides triglycerides
Table 6. The calibrations curves resulting from manual and WorkBench preparation protocols were very similar as shown by the respective slopes and intercepts for each compound. Both preparation methods met the ASTM requirement for correlation coefficient values (r2
) of 0.99 or greater. Manual Prep
Compound slope y-int Glycerin
r2 WorkBench slope y-int r2
Monoolein 1.3446 -0.0171 0.9997 1.3786 0.0044 1.0000 Diolein Triolen
1.0433 0.0028 0.9997 1.1027 0.0049 0.9995 1.2176 -0.0010 0.9999 1.2086 -0.0014 0.9999
0.8303 -0.0018 0.9965 0.8703 0.0030 1.0000 Figure 3. Calibration curves from standards prepared using the WorkBench. Agilent 7890A Series GC
Cool-On-Column Inlet with EPC control Capillary FID with EPC control Agilent 7693A ALS
Area Ratio
0.1 0.2 0.3 0.4 0.5 0.6
0 00 Amount Ratio .2 Area Ratio
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
0 Diolein 350 ug 200 ug 100 ug 50 ug 00.2 0.4 Amount Ratio 500 ug 0.4 Glycerin 50 ug 35 ug 25 ug 15 ug 5 ug
Area Ratio
1.25 1.5 1.75
0.25 0.5 0.75 1
0 100 ug 00 Amount Ratio .5 Area Ratio Triolein
0.1 0.2 0.3 0.4 0.5
0 50 ug 00.2 0.4 Amount Ratio 350 ug 200 ug 100 ug 500 ug 1 Monoolein 1000 ug 750 ug 500 ug 250 ug
Manual Prep
WorkBench Prep
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56
