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A national laboratory of the U.S. Department of Energy

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NREL is operated by Midwest Research Institute ● Battelle Contract No. DE-AC36-99-GO10337 

An Evaluation and Comparison 

of Test Methods to Measure the 

Oxidation Stability of Neat 

Biodiesel 

September 2003 — May 2005 

S.R. Westbrook 

Southwest Research Institute 

San Antonio, Texas 

Subcontract Report 

NREL/SR-540-38983 

November 2005 
An Evaluation and Comparison 

of Test Methods to Measure the 

Oxidation Stability of Neat 

Biodiesel 

September 2003 — May 2005 

S.R. Westbrook 

Southwest Research Institute 

San Antonio, Texas 

NREL Technical Monitor: R. McCormick 

Prepared under Subcontract No. ACE-3-33075-01 

Subcontract Report 

NREL/SR-540-38983 

November 2005 

National Renewable Energy Laboratory

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Operated for the U.S. Department of Energy

Office of Energy Efficiency and Renewable Energy

by Midwest Research Institute • Battelle 

Contract No. DE-AC36-99-GO10337
This publication was reproduced from the best available copy

Submitted by the subcontractor and received no editorial review at NREL

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Acknowledgements 

The author wishes to thank M. S. Voigt, R. H. Ward, J. A. Rios, M. H. Feichtinger, J. A. 

Waynick, and L. M. De Salme for their contributions to this work and the preparation of this 

report. The author also gratefully acknowledges the technical direction of Dr. R. L. McCormick 

and the financial support of the National Renewable Energy Laboratory. This work was 

performed for the U.S. Department of Energy, FreedomCAR and Vehicle Technologies 

Program, Fuels Technologies Subprogram. 

Executive Summary 

Oxidation stability is one of the most important properties of fatty-acid methyl esters (biodiesel) 

used as a blend stock with petroleum diesel to make biodiesel blends. Even so, at the time of this 

writing, there is no oxidation stability requirement in ASTM Standard Specification D6751, the 

specification for biodiesel. The primary reason for the absence of an oxidation stability 

requirement is a lack of agreement on which oxidation stability test method to specify. The 

European biodiesel specification incorporates the Rancimat test as a measure of oxidation 

stability. The Rancimat test is one of the methods under consideration for the ASTM 

specification along with several other stability tests traditionally used for petroleum fuels. 

The purpose of this project was to compare and evaluate several candidate test methods. The 

information gathered from this work will be available to aid in the selection of a test method for 

inclusion in D6751. 

The test methods evaluated during this project were: 

• ASTM1

 D4625, Standard Test Method for Middle Distillate Fuel Storage Stability at 

43°C (110°F). 

• ASTM D6468, Standard Test Method for High Temperature Stability of Distillate Fuels. 

Modified for gravimetric determination of insoluble formation (as described in this 

report). 

• ASTM D2274, Standard Test Method for Oxidation Stability of Distillate Fuel Oil 

(Accelerated Method). Modified, specifically for use with biodiesel, as described later in 

this report. 

• ASTM D3241, Standard Test Method for Thermal Oxidation Stability of Aviation 

Turbine Fuels (JFTOT Procedure). 

• EN 14112, the Rancimat Test, Fat and oil derivatives. Fatty Acid Methyl Ester (FAME). 

Determination Of Oxidation Stability (Accelerated Oxidation Test). 

• Modified IP 306, Determination Of Oxidation Stability Of Straight Mineral Oil. 

• ASTM D5483, Standard Test Method for Oxidation Induction Time of Lubricating 

Greases by Pressure Differential Scanning Calorimetry. 

The test methods were evaluated for their ease of use, applicability to the measurement of 

biodiesel, ability to discern additive effects, and ability to discriminate between biodiesel 

samples of various levels of oxidation stability. 

iii
Two test methods emerged as the most likely choice for inclusion in D6751. They were the 

Rancimat test and a modified version of D2274. For D2274, modification to include collection of 

isooctane insolubles (or other non-polar solvent) may improve precision and accuracy. Other 

modifications to filtration procedures may also be necessary. Additional evaluation of these test 

methods is proposed in order to make a final decision for specification purposes. 

iv
Table of Contents

Acknowledgements........................................................................................................................iii 

Executive Summary.......................................................................................................................iii 

Table of Contents............................................................................................................................ v 

List of Tables ................................................................................................................................. vi 

List of Figures................................................................................................................................ vi 

1.0 Objective ................................................................................................................................... 1 

2.0 Background............................................................................................................................... 1 

2.1 B100 Stability ....................................................................................................................... 2 

2.2 Effect of Anti-Oxidants......................................................................................................... 6 

2.3 Thermal Stability .................................................................................................................. 7 

2.4 Summary............................................................................................................................... 8 

3.0 Procedure .................................................................................................................................. 8 

3.1 Tasks..................................................................................................................................... 8 

3.1.1 Comparative Testing with Unstable Biodiesel .............................................................. 8 

3.1.2 Effect of Antioxidants.................................................................................................... 8

3.1.3 Testing of Commercial Biodiesels................................................................................. 9 

3.2 Test Fuels.............................................................................................................................. 9 

3.3 Test Methods....................................................................................................................... 11 

3.3.1 ASTM D4625............................................................................................................... 11 

3.3.2 ASTM D2274............................................................................................................... 12 

3.3.3 ASTM D6468 at 150°C, 3 hours ................................................................................. 12 

3.3.4 EN 14112 (Rancimat) .................................................................................................. 12 

3.3.5 ASTM D3241, JFTOT Procedure................................................................................ 14 

3.3.6 HPDSC - High Pressure Differential Scanning Calorimeter....................................... 14 

3.3.7 Institute of Petroleum Method 306 (Modified)............................................................ 15 

4.0 Results and Discussion of Data............................................................................................... 15 

4.1 ASTM D4625...................................................................................................................... 15 

4.2 ASTM D2274...................................................................................................................... 16 

4.3 ASTM D6468...................................................................................................................... 21 

4.4 EN 14112 (Rancimat) ......................................................................................................... 22 

4.5 ASTM D3241 (JFTOT) ...................................................................................................... 24 

4.6 HPDSC................................................................................................................................ 25 

4.7 IP Method 306 (Modified).................................................................................................. 26 

4.8 Antioxidant Additives......................................................................................................... 26 

4.9 Oxidation Stability of Commercially Available B100 Fuels.............................................. 28 

4.10 Tests of B20 by D2274 and Rancimat .............................................................................. 29 

5.0 Conclusions on Selection of an Oxidation Stability Test Method for B100........................... 30 

5.1 Test Methods....................................................................................................................... 30 

5.1.1 Rancimat Test .............................................................................................................. 31 

5.1.2 Modified D2274........................................................................................................... 31 

5.2 Specification Limits............................................................................................................ 32 

5.3 Antioxidants........................................................................................................................ 32 

List of Acronyms .......................................................................................................................... 33 

References..................................................................................................................................... 34 

v
List of Tables 

Table 1. Results of Repeated ASTM D2274 Tests at 95°C, 16 hours on Tallow-based Biodiesel 

(Sample AL-25954)........................................................................................................................ 6 

Table 2. Results of D6468 Using Both Standard And Modified Methods for Three Biodiesel 

Fuels (B100) And Two Petroleum Diesel Fuels a

........................................................................... 8 

Table 3. Test Fuels.......................................................................................................................... 9 

Table 4. Characterization Data for Test Fuels.............................................................................. 10 

Table 5. Fatty Acid Methyl Ester Content of Test Fuels (As Measured by GCMS).................... 11 

Table 6. Results of ASTM D4625 Tests....................................................................................... 16 

Table 7. Effect of Antioxidant at Different Temperatures on total insolubles (mg/100 ml) in the 

AST D2274 test (data from Stavinoha and Howell, reference 2)................................................. 18 

Table 8. Results for Testing of B100 by Modified D2274 (95°C)................................................ 20 

Table 9. Modified D2274 (110°C) of B100 and B100 plus Antioxidant...................................... 20 

Table 10. Modified D2274 – Polymer Solubility ......................................................................... 21 

Table 11. Results for D6468 Tests................................................................................................ 21 

Table 12. Rancimat Test Results .................................................................................................. 23 

Table 13. Results of Testing B100 Samples by ASTM D3241 (JFTOT)..................................... 25 

Table 14. HPDSC Results for Biodiesel Plus Antioxidants (Temperature Ramp = 10°C/min)... 26 

Table 15. Results for Testing of B100 by Modified IP 306 ......................................................... 26 

Table 16. Description of Additives Used...................................................................................... 28 

Table 17. Effect of Food-Grade Antioxidants -- Rancimat Testing ............................................. 28 

Table 18. Effect of Petroleum Antioxidants -- Rancimat Testing ................................................ 28 

Table 19. Rancimat Test Results for B100 Fuel Quality Survey Samples................................... 29 

Table 20. Rancimat Results for Samples of Commercially Available B100 Fuels from Various 

Sources.......................................................................................................................................... 29 

Table 21. Rancimat Test (110°C) Results for B20 Blends........................................................... 29 

List of Figures 

Figure 1. Results Of Long-Term Storage Stability Testing For Several B100 Biodiesel Samples 

Via ASTM D4625 (43°C). .............................................................................................................. 3 

Figure 2. Results Of Accelerated Oxidation Stability Testing For B100 Samples Using ASTM 

D2274 At Different Times And Temperatures. .............................................................................. 4 

Figure 3. Results Of Accelerated Oxidation Stability Testing For B100 Samples Using ASTM 

D2274 At Different Times And Temperatures. .............................................................................. 5 

Figure 4. Results Of Accelerated Stability Determination By ASTM D2274 At 80°C For 24 

Hours For CL00-289 Treated With Several Antioxidants.............................................................. 7 

Figure 5. Detailed Requirements for Biodiesel per ASTM D6751 ................................................ 9 

Figure 6. Rancimat Instrument Connected to Notebook Computer. The Standard Rancimat Can 

Analyze Eight Samples at One Time ............................................................................................ 13 

Figure 7. Typical Rancimat Test Data Trace for a Sample with an Induction Period of 4.84 Hours

....................................................................................................................................................... 14 

vi
Figure 8. D2274 Time and Temperature Study Results ............................................................... 19 

Figure 9. Thermal Stability D6468, Test Results for B100 and Petroleum Diesel Samples (See 

Reference 9).................................................................................................................................. 22 

Figure 10. Arrhenius Plot of Rancimat Induction Periods at Selected Temperatures.................. 24 

Figure 11. JFTOT Tube-Deposit Volume versus Temperature .................................................... 25 

Figure 12. Antioxidant Efficacy for Three Food-Grade Additives via Rancimat at 110°C, soy 

based biodiesel.............................................................................................................................. 27 

Figure 13. Comparison of Antioxidant Effects for Selected Petroleum and Food-Grade 

Additives, Rancimat at 110°C....................................................................................................... 27 

vii
1.0 Objective 

The objectives of this project are to compare several stability test methods using biodiesel typical 

of the U.S. market: 

a. Using the currently accepted tests (D2274 and EN14112) as well as other tests that might 

provide additional or more accurate information. 

b. To examine the effect of common anti-oxidants. 

c. Verify these results using a subset of the test methods on commercial biodiesel samples 

produced from soybean oil and yellow grease. 

2.0 Background 

An important goal of the United States Department of Energy (US DOE) Fuels Technologies 

Program is to develop renewable diesel blending-components in order to displace imported 

petroleum and to reduce emissions of global warming gases. Biodiesel is an important renewable 

diesel blending component. Because of its chemical structure, biodiesel can be very sensitive to 

oxidative and thermal degradation. The sensitivity varies depending on the feedstock and the 

presence of naturally occurring antioxidants in the finished fuel. The oxidation stability 

characteristics of biodiesel can also be improved through the use of commercially available 

antioxidant additives. Oxidation can lead to the formation of corrosive acids and deposits that 

may cause increased wear in engine fuel pumps and fuel injectors. 

ASTM International (American Society of Testing and Materials, ASTM) Standard Specification 

D6751 is the quality specification for neat (100%) biodiesel or B100 that is to be used for 

blending with petroleum diesel. This specification does not currently contain a requirement for 

fuel stability. The lack of a stability requirement in D6751 is a major barrier to increased 

acceptance of biodiesel by engine and fuel injection equipment manufacturers, and thus to 

increased markets for this fuel. In order to include such a requirement, a suitable test method 

must first be identified, as well as possible alternate test methods. Presently available stability 

tests have been developed for petroleum-derived fuels. Biodiesel is chemically different enough 

that these tests do not produce meaningful results. Under this study basic data on how biodiesel 

is oxidatively and thermally degraded under the conditions of various stability test methods was 

acquired. These data will point to appropriate test method modifications, and ultimately lead to 

improved tools for studies of biodiesel stability and improved stability test methods for inclusion 

in standard specifications. 

Prior work under National Renewable Energy Laboratory (NREL) subcontract number ACG-7-

17066-01 has shown that a modified version of ASTM Test Method D2274 may distinguish 

between stable and unstable B100 samples in some cases. 

In this earlier study, results from the long term D4625 storage test were compared to results of an 

accelerated stability test based on ASTM D2274. D2274 was modified via the following

procedures: 

1
• Use Whatman type GF/F glass fiber filters (because of incompatibility of cellulose ester

filters with biodiesel) 

• Retain aged and filtered fuel and analyze it for viscosity and total acid number 

The results of the study were used to determine appropriate time and temperature for aging. 

Thermal stability was examined using ASTM D6468 (also called the DuPont F21 test) modified 

to include filtration and gravimetric analysis for insolubles. 

2.1 B100 Stability 

For comparison of long-term fuel storage and accelerated oxidation tests the following fuels 

were employed: 

• CL00-288 (soy-biodiesel) 

• CL00-289 (soy-biodiesel) 

• CL00-290 (a 50/50 blend of CL00-288 and CL00-289) 

• AL-25842 (a soy-biodiesel treated with 200 ppm of Tenox-21 antioxidant) 

Results of ASTM D4625 for 16 weeks are shown in Figure 1. With the exception of the Tenox21 treated fuel, all fuels can be considered unstable based on either insoluble formation or acid 

formation during long term storage. There may be some question regarding the insolubles data 

for CL00-289 as an increase followed by a decrease was observed, but this fuel was clearly 

unstable based on acid number. All of the fuels would meet the viscosity requirements of D6751, 

although CL00-289 and CL00-290 exhibited significant increases. 

Based on these results it is clear that no one measure of instability (i.e. insolubles, acid value, or 

viscosity) will be adequate for measuring or predicting stability of B100 fuels. At a minimum, 

measurement of insolubles and either acid value or viscosity will be required. 

The fuels tested above were also tested using ASTM D2274 over a range of times and at three 

different temperatures. The objective of these tests was to select a time and temperature that

distinguishes between stable and unstable fuels. Results are summarized in Figure 2 for the three 

unstable fuels. One approach to selecting an appropriate accelerated oxidation test is to use the 

mildest conditions (lowest temperature and shortest time) that reveal instability. For fuel CL00-

288 instability is only revealed at 95°C and for times of 16 hours or longer, suggesting that for 

this set of fuels these are the required conditions. Figure 3 shows a similar data set for the Tenox21 treated fuel (AL-25842) as well as for a tallow-based biodiesel (AL-25954) and a yellowgrease-based biodiesel (AL-25843). All test conditions predict the Tenox-21 treated fuel to be 

stable. Both the tallow- and yellow grease-based biodiesels are predicted to be unstable using the 

95°C/16 hr test condition, as well as at milder test conditions. Based on these results the 

selection of 95°C and 16 hours would appear to be appropriate for the ASTM D2274 test. 

The tallow-based sample was analyzed using these test conditions to provide a preliminary 

measure of test repeatability and results are listed in Table 1. The coefficient of variation for 

these measurements is roughly 13% for insolubles and acid number, and 1% for viscosity. For 

this sample the insoluble, acid, and viscosity levels rose well above the upper limits in D6751 

2
and thus repeatability is adequate for determining stability. For samples that are less obviously 

unstable improved repeatability may be required. 

In summary, the results of oxidative stability testing for B100 samples indicate that the D2274 

test can predict if a fuel is stable or not. The most appropriate testing conditions are 95°C for 16 

hours. However, additional method development for determination of the mass of insolubles and 

for determination of acid number may be needed in order to reduce experimental error to 

acceptable levels. 

Tot

al A

cid Number, mg KOH/g

0.0

0.5

1.0

1.5

2.0

2.5

3.0

CL00-288

CL00-289

CL00-290

AL-25842

Insoluble, mg/100 ml

0

1

2

3

4

5

CL00-288

CL00-289

CL00-290

AL-25842

Week

0 2 4 6 8 10 12 14 16 18

Visc

o

sity

@

4

0

oC, c

St 3.5

4.0

4.5

5.0

5.5

6.0

6.5

CL00-288

CL00-289

CL00-290

AL-25842

Figure 1. Results Of Long-Term Storage Stability Testing For Several B100 Biodiesel Samples Via 

ASTM D4625 (43°C). 

3
CL00-288

Time, hours

0 5 10 15 20 25 30

Ins, TAN, or Vis

0246

Insolubles, mg/100 mlTotal Acid Number, mg KOH/gViscosity at 40C, cSt

CL00-289

Time, hours

0 5 10 15 20 25 30

Ins, TAN, or Vis

0246

CL00-290

Time, hours

0 5 10 15 

Ins, TAN, or Vis

0246

80oC

CL00-288

Time, hours

0 5 10 15 20 25 30

Ins, TAN, or Vis

0246

CL00-289

Time, hours

0 5 10 15 20 25 30

Ins, TAN, or Vis

0246

CL00-290

Time, hours

0 5 10 15 

Ins, TAN, or Vis

0246

87oC

CL00-288

Time, hours

0 5 10 15 20 25 30

Ins, TAN, or Vis

0246

CL00-289

Time, hours

0 5 10 15 20 25 30

Ins, TAN, or Vis

0246

CL00-290

Time, hours

0 5 10 15 

Ins, TAN, or Vis

0246

95oC

Figure 2. Results Of Accelerated Oxidation Stability Testing For B100 Samples Using ASTM D2274 At Different TimTemperatures. 

4
AL-25842

Time, hours

0 2468 10 12 14 16 18

Ins, TAN, or Vis

0246

Insolubles, mg/100 mlTotal Acid Number, mg KOH/gViscosity at 40C, cSt

AL-25954

Time, hours

0 2468 10 12 14 16 18

Ins, TAN, or Vis

0246

AL-25843

Time, hours

0 5 10 15 

Ins, TAN, or Vis

0246

80oC

AL-25842

Time, hours

0 2468 10 12 14 16 18

Ins, TAN, or Vis

0246

AL-25954

Time, hours

0 2468 10 12 14 16 18

Ins, TAN, or Vis

0246

AL-25843

Time, hours

0 5 10 15 

Ins, TAN, or Vis

0246

87oC

AL-25842

Time, hours

0 2468 10 12 14 16 18

Ins, TAN, or Vis

0246

AL-25954

Time, hours

0 2468 10 12 14 16 18

Ins, TAN, or Vis

0246

AL-25843

Time, hours

0 5 10 15 

Ins, TAN, or Vis

0246

95oC

Figure 3. Results Of Accelerated Oxidation Stability Testing For B100 Samples Using ASTM D2274 At Different TimTemperatures. 

5
Table 1. Results of Repeated ASTM D2274 Tests at 95°C, 16 hours on Tallow-based 

Biodiesel (Sample AL-25954) 

Run Total Insolubles,

mg/100 ml 

Total Acid Number, 

mg KOH/g 

Viscosity @40°C, 

cSt 

1 7.77 6.97 6.43

2 5.92 5.77 6.32

3 6.55 5.10 6.24

4 7.80 5.57 6.27

Mean 7.010 5.853 6.315

Standard Deviation 0.9312 0.7962 0.0835 

Coefficient of Variation 13.3% 13.6% 1.32%

95% conf 1.4814 1.2666 0.1328 

Upper Limit* 0.05 0.8 6.0 *

Upper limit for these properties based on specifications in D6751 for B100. Runs 1 and 

2 from SwRI report for Task 8A, November 23, 1999. Runs 3 and 4 from SwRI report to 

NBB dated August 3, 2001. 

2.2 Effect of Anti-Oxidants 

A task was conducted to determine if ASTM D2274 at 80°C and for 24 hours would 

reveal the effect of antioxidants on stability for fuel CL00-289. While subsequent results 

suggest that 80°C is not the best temperature for this test, it was selected based on results 

of another study2

 that indicated lower temperatures were required to reveal the impact of 

antioxidants. Additionally, for this fuel a temperature of 80°C was adequate to reveal 

instability in the accelerated test. The test fuels were: 

• CL00-289 

• CL00-289 plus 200 ppm BHT 

• CL00-289 plus 200 ppm TBHQ 

• CL00-289 plus 1000 ppm Tenox-21 

Figure 4 shows the results for total insolubles, acid number, and viscosity following the 

modified D2274 procedure for these fuels. The unadditized fuels are again predicted to be 

unstable by this test. The addition of 200 ppm of BHT is very effective at stabilizing the 

fuel under these conditions with essentially no change in acid number or viscosity and 

only a small formation of insolubles. Both TBHQ and Tenox-21 improve stability 

substantially. 

6
Total In

s

olubles, mg/d

L

Acid Value, mg KOH/g

Viscosit

y

@

4

0

C, cSt

0

2

4

6

8

10

12

Total Insolubles

Acid Value 

Viscosity

Initial

Values

Unadditized

Base Fuel

BHT

TBHQ

Tenox-21

Figure 4. Results Of Accelerated Stability Determination By ASTM D2274 At 80°C For 24 

Hours For CL00-289 Treated With Several Antioxidants. 

It is interesting to note that in this case 1,000 ppm of Tenox-21 did not produce a 

completely stable fuel, while the results shown above in Figure 1 indicate that only 200 

ppm of Tenox-21 was adequate to accomplish this in the long term test (ASTM D4625). 

It seems clear that additional test data are required to understand the impact of 

antioxidants in the accelerated test. More specifically, it is desirable to test an unstable

biodiesel over a range of antioxidant blending levels and temperatures using several food 

and refinery antioxidants. 

2.3 Thermal Stability 

Thermal stability of three B100 samples and two petroleum diesel samples was assessed 

using ASTM D6468 (duPont F21 test). Both the standard version of D6468 and a 

modified version were used. In the standard version the fuel is filtered following high 

temperature exposure and the reflectance of the filter is measured. Reflectance decreases 

with increasing levels of insolubles. A modified method where insolubles are determined 

gravimetrically was also used. Table 2 reports results for both approaches. Note that none 

of the B100 fuels showed any appreciable degradation under any of the test conditions. 

7
Table 2. Results of D6468 Using Both Standard And Modified Methods for Three Biodiesel 

Fuels (B100) And Two Petroleum Diesel Fuels a

90 minutes 180 minutes 360 minutes

Sample % 

Reflectance 

Gravimetric 

mg/dL 

% 

Reflectance 

Gravimetric 

mg/dL 

% 

Reflectance 

Gravimetric 

mg/dLb

CL00- 93.10 -0.80 93.40 -1.20 91.40 -0.09

288 98.00 -0.26 90.00 0.06 91.00 -1.60

CL00- 99.70 -0.09 99.40 -0.23 98.70 -0.34

289 99.60 -0.23 99.40 -0.54 99.30 -0.63

CL00- 95.90 -0.03 95.80 0.03 94.20 -0.63

290 94.70 -0.91 95.70 -0.17 93.10 -1.06

AL- 7.50 1.86 4.10 1.91 2.90 3.09

25844 15.70 1.00 3.80 2.03 2.60 2.80

AL- 4.10 5.60 2.80 6.23 1.80 9.80

22662 4.10 5.83 2.80 7.51 1.90 10.00 a

AL-25844 is a stable petroleum diesel and AL-22662 is an unstable petroleum diesel. 

b

Negative values indicate that the filter lost mass and are an indication of the error of this 

test. 

2.4 Summary 

The results of this prior study clearly show the potential of biodiesel to exhibit oxidation 

instability in different environments. As a follow on to this work, the current study was 

conducted with the goal of comparing a wider range of test methods for use in measuring 

biodiesel stability and the effect of antioxidants. 

3.0 Procedure 

3.1 Tasks 

3.1.1 Comparative Testing with Unstable Biodiesel 

The objective of this task is to compare results using a variety of oxidative and thermal 

stability test methods using two highly characterized unstable biodiesels. Based on these 

results, a more limited set of tests and conditions were selected for further examination. 

The following subtasks were also performed: 

3.1.2 Effect of Antioxidants 

The objective of this task is to examine the performance of common anti-oxidant 

chemistries as well as the sensitivity of selected test methods to the effects of these 

additives. 

8
3.1.3 Testing of Commercial Biodiesels 

This task will determine if what was learned in the above two tasks can be generalized to 

other soy-based biodiesels (containing natural antioxidants), as well as biodiesels 

produced from waste cooking oil and animal fat. 

3.2 Test Fuels 

The test fuels are listed in Table 3. Neither test fuel contained any additives as received. 

The fuels were tested against ASTM Standard Specification D6751. The detailed 

requirements of the specification are shown in Figure 5. Inspection test results are listed 

in Table 4. The test fuels meet all requirements with the exception of the acid number for 

soy-derived fuel. The limit on acid number is 0.8 mg KOH/g. The measured value of 1.46 

indicates that some oxidation of this material has already begun to occur. 

Table 5 gives the results of Fatty Acid Methyl Ester (FAME) analysis for the two B100 

test fuels. Also contained in the table is the typical composition for soy-derived B100. It 

is not meaningful to give typical results for yellow grease since it is derived from

numerous different feedstocks. The FAME analysis indicates that the soy-derived test 

fuel is atypical of soy biodiesel with 60% oleic acid (C18: 1) and almost no C18: 3. 

Nevertheless, this material contained very low levels of natural antioxidants3

 and was not 

found to be particularly stable on various stability tests. 

Table 3. Test Fuels 

Laboratory Identification Description 

27007 -- Soy (CL03-842) Distilled Soy Methyl Ester Biodiesel 

27008 -- Yellow Grease (CL03-843) Low Sulfur, Yellow Grease Biodiesel 

Figure 5. Detailed Requirements for Biodiesel per ASTM D6751

9
Table 4. Characterization Data for Test Fuels 

Property Soy Yellow Grease

Copper Strip Corrosion, D130 1A 1A 

Water and Sediment, vol%, D2709 < 0.005 <0.005

Flash Point, °C, D93 172 176

Kinematic Viscosity @ 40°C, mm2/s, D445 4.72 4.64

Cloud Point, °C, D2500 8 13

Cetane Number, D613 65 64 

Sulfated Ash, D874, mass% <0.01 <0.01 

Carbon Residue, D4530, mass% 0.02 0.007 

Free Glycerin, D6584, mass% 0.007 0.008 

Total Glycerin, D6584, mass% 0.051 0.063 

Phosphorus Content, D4951, ppm <5 <5 

Water and Sediment, D2709, volume% <0.005 <0.005

Total Sulfur, D5453, ppm <5 6.7 

Total Acid Number, D664, mg KOH/g 1.46 0.59 

Vacuum Distillation, D1160, °C 

 IBP 

 10% 

 50% 

 90% 

 99% 

 Residue 

 Loss 

362 

381 

381 

386 

435 

0.5 

348 

380 

401 

408 

422 

1 

10
Table 5. Fatty Acid Methyl Ester Content of Test Fuels (As Measured by GCMS)

4

FAME Name Soy, mass %

Typical Soy Test Fuel 

Yellow Grease, 

mass %

C12:0 Methyl Laurate

C14:0 Methyl Myristate 0 0.17 0.22 

C14:1 0 ---

C15:0 0 ---

C16:0 Methyl Palmitate 12 14.21 12.26 

C16:1 Methyl Palmitoleate 0 ---

C16:2 0 ---

C17:0 0 ---

C17:1 0 ---

C18:0 Methyl Stearate 4 12.02 5.70 

C18:1 Methyl Oleate (9) 23 58.24 30.27 

C18:2 Methyl Linoleate (9,12) 55 13.56 47.69 

C18:3 Methyl Linolenate (6,9,12) 7 0.94 3.28 

C20:0 Methyl Arachidate 0.68 0.32 

C22:0 Methyl Behenate 0.09 0.27 

C24:0 Methyl Lignocerate

1 

0.09 

3.3 Test Methods

Although there is an oxidation stability requirement in the European biodiesel 

specification (EN 14214), there is not one in the ASTM specification. The results of this 

project will be used as part of the selection of a test method for the ASTM specification. 

Several candidate test methods were selected for evaluation, as follows: 

3.3.1 ASTM D4625 

Standard Test Method ASTM D4625 is the most widely accepted test method for 

assessing the storage stability of middle distillate, petroleum fuels. The fuel is stored at 

43°C for selected periods up to 24 weeks. One week of storage in this test is generally 

accepted as equivalent to one month of storage at 17°C (65°F). Typically a sample is 

filtered to determine total insolubles on a weekely basis. For testing of biodiesel 

Whatman GF/F filters (47 mm diameter) were used. After filtering the aged sample the 

filtrate was analyzed for total acid number (ASTM D664) and kinematic viscosity at 

40°C (ASTM D445). 

As an additional analysis, 100 ml of the aged, filtered fuel was mixed with 400 ml of isooctane to precipitate any polar polymeric materials that may have formed as a result of

ageing. The aged fuel/iso-octane mixture was filtered through a separate pair of filters. 

This was done to investigate a previous study that concluded that ageing of B100 under 

these conditions does not result in the formation of insolubles because the oxidized and 

polar polymers that are formed are soluble in the biodiesel. Dilution with a non-polar 

solvent, such as iso-octane, was shown to result in precipitation of these polymers. 5

11
3.3.2 ASTM D2274 

A 350 ml volume of filtered middle distillate fuel is aged at 95°C (203°F) for 16 h. 

Oxygen is bubbled through the sample at a rate of 3 L/h. After aging, the sample is 

cooled to approximately room temperature before filtering to obtain the filterable 

insolubles quantity. Adherent insolubles are then removed from the oxidation cell and 

associated glassware with trisolvent (a mixture of equal parts toluene, acetone, and 

methanol). The trisolvent is evaporated to obtain the quantity of adherent insolubles. The 

sum of the filterable and adherent insolubles, expressed as milligrams per 100 ml, is 

reported as total insolubles. As with the D4625 analyses, 100 ml of the aged, filtered fuel 

was mixed with 400 ml of iso-octane and filtered. 

Whatman GF/F filters (47 mm diameter) were used. 

After filtering the aged sample, but prior to solvent washing of the filters, an aliquot of

the filtrate was obtained for additional testing. The aliquot was analyzed for total acid 

number (ASTM D664) and kinematic viscosity at 40°C (ASTM D445). 

As an additional analysis for biodiesel-soluble polymers, 100 ml of the aged, filtered fuel 

was mixed with 400 ml of iso-octane. The aged fuel/iso-octane mixture was also filtered 

through a separate pair of filters. 

3.3.3 ASTM D6468 at 150°C, 3 hours 

Two 50-mL volumes of filtered middle distillate fuel were aged for 90 or 180 minutes at 

150°C in open tubes with air exposure. After aging and cooling, the fuel samples were 

filtered and the average amount of filterable insolubles is estimated by measuring the 

light reflectance of the filter pads. An unused filter pad and a commercial black standard 

define the 100 and 0 % extremes of the reflectance rating range, respectively. 

The reflectance measurement works well with petroleum diesel fuel because the particles 

formed during the aging are typically brown or black. Hence, increased amounts of 

particles result in decreased reflectance. With biodiesel, the particles and polymers 

formed have almost no visible color. Therefore, increased amounts of biodiesel 

particles/polymers result in little or no change in reflectance. This means that the test

gives best results when the biodiesel particles/polymers are measured gravimetrically. 

3.3.4 EN 14112 (Rancimat) 

The Rancimat test instrument is pictured in Figure 6. This is the required stability method 

in the European biodiesel specification. The induction period prior to the onset of rapid 

oxidation is measured. 

A stream of purified air is passed through the sample, which is heated to a specified 

temperature. The gases released during the oxidation process, together with the air, are 

12
passed into a flask containing deionized water. The flask also contains an electrode to 

measure conductivity. The electrode is connected to a measuring and recording device. 

The time at which the conductivity begins to increase rapidly is defined as the induction 

time. Acids produced during the oxidation process and absorbed in the water cause this 

accelerated increase. The volatile acids are primarily formic acid, but also include lower 

levels of acetic and other acids.6

 A plot of a typical Rancimat test is shown in Figure 7. 

This figure shows the deionized water conductivity versus time for the Rancimat test. 

For this particular sample conductivity remains unchanged or very low for roughly 4.5 

hours. At this point in time the conductivity of the water begins to increase rapidly 

because of oxidation of the biodiesel sample yielding volatile acids that are carried by the 

flowing air into the water bath. The first derivative of the conductivity curve shows the 

inflection point (maxima in the first derivative) that defines the induction time. Recent 

results show that Rancimat induction times correlate well with induction times measured 

by ASTM D525, Standard Test Method for Oxidation Stability of Gasoline.7

Figure 6. Rancimat Instrument Connected to Notebook Computer. The Standard Rancimat 

Can Analyze Eight Samples at One Time 

13
 4.84 

0

5

10

15

20

25

30

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

μS/c

m

h

Conductivity 

First Derivative of 

Conductivity 

Figure 7. Typical Rancimat Test Data Trace for a Sample with an Induction Period of 4.84 

Hours 

3.3.5 ASTM D3241, JFTOT Procedure 

This test method for measuring the high temperature stability of gas turbine fuels uses the 

Jet Fuel Thermal Oxidation Tester (JFTOT) that subjects the test fuel to conditions that 

can be related to those occurring in gas turbine engine fuel systems. The fuel is pumped 

at a fixed volumetric flow rate through a heater after which it enters a precision stainless 

steel filter where fuel degradation products may become trapped. For the work in this 

project, the precision filter had to be removed because it plugged too quickly. The 

apparatus uses 450 mL of test fuel ideally during a 2.5 h test. The essential data derived 

are the amount of deposits on an aluminum heater tube as determined by ellipsometry (an 

optical technique for measuring the thickness of thin films), and the rate of plugging of a 

17μ nominal porosity precision filter located just downstream of the heater tube. 

3.3.6 HPDSC - High Pressure Differential Scanning Calorimeter 

These tests were conducted using a modification of ASTM D5483, Standard Test Method 

for Oxidation Induction Time of Lubricating Greases by Pressure Differential Scanning 

Calorimetry. A small quantity of sample is weighed into a sample pan and placed in a test 

cell. The cell is heated to a specified temperature and then pressurized with oxygen to 

500 pounds per square inch. The cell is held at a regulated temperature and pressure until 

an exothermic reaction occurs. The extrapolated onset time is measured and reported as 

the oxidation induction time for the sample under the specified test temperature. 

14
3.3.7 Institute of Petroleum Method 3068

 (Modified) 

This test method is similar to D2274. The sample size is only 25 grams as opposed to 350 

mL for D2274. The oxidation tubes are correspondingly smaller. Dry oxygen is passed 

into the reaction tubes (containing the B100) for 16 hours. The IP 306 method calls for an 

oxidation catalyst (copper wire) in one sample during aging but no catalyst was used in 

any of the tubes during this testing. The heating block is held at a temperature of 120°C. 

The volatile acids driven off by the flowing oxygen were absorbed in de-ionized water. 

The volatile and oil-soluble acidities produced in the B100 were measured at the end of 

the test. The insoluble polymers were also measured gravimetrically. A solvent of equal 

parts toluene, acetone, and methanol was used in place of chloroform wherever it was

called for in the test method. 

IP 306 is a viable alternative test method to D2274 although more work has been done 

with D2274. The largest disadvantage of IP 306 is the small sample size. Based on 

experience with other test methods (such as D6217, D2276, and D2274) it is very likely 

that the 25 g sample size significantly reduces the sensitivity of the polymer 

measurement. 

4.0 Results and Discussion of Data

The results of analyses with each test are discussed below. 

4.1 ASTM D4625 

The results for the D4625 test are presented in Table 6. In general, each of the properties 

showed an increase with storage time, as expected. The degree of change varied with the 

fuels. This is in agreement with an earlier project, which found that D4625 is sensitive to 

stability differences in the fuels.9

 While the soy biodiesel tested here exhibited an 

atypical fatty acid composition, results for this test are similar and in line with what has 

been observed previously for relatively unstable soy biodiesel (see Figure 1). Note that 

the yellow grease sample had a significant increase in iso-octane insoluble material 

(dissolved polymers) between weeks 4 and 8. Yet, there was almost no change in the 

viscosity of the fuel. This indicates that perhaps viscosity is not sufficiently sensitive to 

changes in polymer formation. Also, the conditions of this test are very mild compared to 

some of the other test methods often used to measure oxidation stability. This test is more 

representative of changes in the biodiesel during longer periods of quiescent storage, 

typically at temperatures below 30°C. It is not representative of the higher temperatures 

and greater oxygen exposure found in a diesel vehicle fuel system. 

Although it is a generally accepted test for the storage stability of petroleum fuels, similar 

correlations to the storage of biodiesel have yet to be demonstrated. Until those 

correlations have been demonstrated, the ability of this test to measure storage stability is 

unclear. The milder conditions and very long test times used in this method make it a 

poor choice as a convenient and predictive measure of oxidation stability. 

15
Table 6. Results of ASTM D4625 Tests

Biodiesel Weeks

Total 

Insolubles 

(mg/L) 

Total Acid 

Number 

(mg KOH/g) 

Kinematic 

Viscosity @ 

40°C, mm2/s

iso-octane 

Insoluble Material

(mg/L) 

4 3.0 1.6 5.0 0.1

8 0.3 1.9 5.2 4.2

Soy 

12 1.7 2.5 5.4 2.8

4 0.1 0.7 4.7 3.8

8 0.4 0.7 4.7 2.8

Yellow 

Grease 

12 7.5 0.9 4.8 12.2

4.2 ASTM D2274 

The D2274 test has been used in various, non-ASTM, diesel fuel specifications for over 

30 years. During that entire period, numerous researchers have spent countless hours, and 

dollars, attempting to improve the precision of the method and determine its correlation 

to long-term storage. Many of the sources of imprecision were identified and corrected. 

Still, the published precision for the method is less than most users consider acceptable 

(see below for published precision). 

Precision and Bias Statement from ASTM D2274-03a 

For petroleum diesel fuel, it has been demonstrated that there is no correlation between 

the results of D2274 and long-term storage under normal conditions. The general 

consensus is that the test can discriminate between very stable fuels and very unstable 

16
fuels. The marginal fuels tend to give mixed results compared to long-term storage. It is 

assumed that the poor correlation is because the high test temperature and constant

oxygen replenishment initiate reactions that would not normally occur at the lower 

temperatures and quiescent storage. 

The test conditions that make D2274 a poor predictor of storage stability are the very 

conditions that make the test a promising candidate for measuring biodiesel oxidation 

stability. Because the chemistry of biodiesel degradation is almost entirely oxidation 

chemistry, higher temperatures and constant oxygen replenishment are realistic 

parameters for a biodiesel test method. Indeed, all the test methods that have either been 

adopted or seriously considered have these parameters in common with D2274. 

Most of the previous work with D2274 as a test for biodiesel has centered on selection of 

an appropriate test temperature and test time. The standard temperature is 95°C and the 

standard test time is 16 hours. More recently, some researchers have begun to study the 

solubility factor of biodiesel. This issue was identified and first studied under the 

European Biostab project. Under that project, studies of stability test methods produced 

the following as one of the observations from the work: 

“...no significant insoluble formation was observed. The conclusion was that 

polymer formed during storage of biodiesel in controlled conditions are soluble in 

oxidised biodiesel, thanks to its high polarity and become insoluble only when 

oxidised biodiesel is mixed with diesel fuel...” 

It is now understood that the solubility factor plays a significant role in the oxidation 

stability of biodiesel. Also, it is now assumed that test methods such as D2274, which 

measure insolubles formation, must also incorporate a measurement of the polymers that 

are insoluble when the B100 is mixed with a non-polar solvent. 

As such, consideration of D2274 for biodiesel oxidation stability means selection of three 

parameters: 

1. Test temperature. 

2. Test time. 

3. Ratio of aged B100 to non-polar solvent for measuring solubility effects. 

Of the three parameters listed above, items 1 and 2 have been studied in greatest detail. 

Test temperatures from 80°C to 110°C have been proposed and studied. The results are 

mixed and no clear choice stands out. Stavinoha and Howell showed data that indicated 

temperatures above 80°C could mask the effects of antioxidants. This conclusion was 

based on very limited data (shown in Table 7). However, it is a significant observation 

and is deserving of further study. The effects of test temperatures above 110°C are not yet 

known but temperatures that high have been known to decompose natural inhibitors.10

17
Table 7. Effect of Antioxidant at Different Temperatures

on total insolubles (mg/100 ml) in the AST D2274 test 

(data from Stavinoha and Howell, reference 2) 

BHT Conc., ppm B100 (1) 80°C B100 (1) 95°C 

0 17.2 12.1

120 8.1 16.5

240 1.2 16.5

Test times ranging from 4 hours to 8 hours have been evaluated. Since there is no 

established correlation of this test with long-term storage and no data with which to draw 

such a correlation, selection of a test time rests on other criteria. The main criteria are 

ease of use and sufficient time to allow discrimination between fuels of various qualities. 

For the purposes of a specification test/quality control, shorter test times are desirable. 

Longer test times typically provide more information on the degradation of the sample. In 

a previous study, Westbrook and Stavinoha examined a large matrix of test times and 

temperatures. A plot of total insolubles data from that study is shown in Figure 8. As 

would be expected, the best discrimination between fuels comes at higher temperatures 

and longer test times. These experiments did not, however, include an examination of 

solubility effects or antioxidant additives. As such, it is difficult to make test condition 

selections on those results. 

18
0 5 10 15 20 25

0

5

10

15

20

25

30

0 5 10 15 20 25

-10

-5

0

5

10

15

20

25

1-B

1-B

2-B

2-B

3-B

3-B

Hours

0 5 10 15 20 25

Total Ins

olubles, mg/d

L

0

5

10

15

20

25

30

80oC

87o

C

95o

C

Figure 8. D2274 Time and Temperature Study Results 

For the purposes of the current study, we ran tests at 95°C and 110°C. The focus of this 

work however, was on the solubility factor rather than test temperature or test duration. 

The data presented in Table 8 are for the fuels tested by the modified D2274 at 95°C. The 

data presented in Table 9 are for the modified D2274 conducted at 110°C. From the data 

in these two tables, it is interesting to note the difference in the amount of iso-octane 

insolubles at the two temperatures. Note that each of the fuels tends to form similar 

amounts of total insolubles, irrespective of the temperature. The amounts of iso-octane 

insolubles, however, differ significantly, depending on the temperature. For each of the 

fuels, the higher test temperature results in higher levels of iso-octane insoluble polymers. 

19
This demonstrates the significant effect that the solubility of B100 can have on the 

amount of measured total insolubles. These results are especially significant when one 

remembers that most biodiesel is used in a blend with petroleum diesel fuel, a 

significantly less polar solvent than B100. And, we can expect that this solubility 

difference will grow as more ultra-low sulfur diesel fuel appears in the marketplace as 

removal of heteroatoms and aromatics will make this fuel a less polar solvent. These 

results support the inclusion of a measurement of iso-octane insolubles in any oxidation 

test that measures insolubles formation. 

Table 8. Results for Testing of B100 by Modified D2274 (95°C) 

Biodiesel Total Insolubles

(mg/100 ml) 

Total Acid 

Number 

(mg KOH/g) 

Kinematic 

Viscosity @ 

40°C, mm2

/s

iso-octane Insoluble 

Material (mg/100 ml) 

Soy 7.7 7.1 6.6 2.1

Yellow 

Grease 

4.1 1.5 5.3 1.4

Table 9. Modified D2274 (110°C) of B100 and B100 plus Antioxidant 

 mg/100 ml 

Sample Tube Filt. Insols Adh. Insols Tot. Insols Iso-octane Insols 

Soy 1 0 3.4 3.4 19.4

 2 10.7 2 12.7 15.7 

Soy + 500 ppm TBHQ 1 0 2.8 2.8 16.1 

 2 7.9 2.1 10 18.2 

Soy + 500 ppm Pet AO 203 1 17.3 1.9 19.2 15.3 

2 19.7 3.2 22.9 13.2

Yellow Grease 1 0 2.2 2.2 48.0 

 2 0 2.6 2.6 40.2 

Yellow Grease + 500 ppm 

TBHQ 1 0 1.9 1.9 38.2

 2 0 1.5 1.5 35.8 

At this higher temperature, the soy-derived B100 tended to form more total insolubles but 

less iso-octane insolubles as compared to the yellow grease B100. Also, there seems to be 

little measurable effect of addition of antioxidant. The only exception is the marked 

increase in filterable insolubles when the petroleum antioxidant was added. 

One interesting observation from these D2274 results is presented in Table 10. Note that 

raising the test temperature from 95°C to 110°C had almost no effect on the measured 

total insolubles but a very marked effect on the amount of iso-octane insolubles. These 

data further demonstrate the significant ability of B100 to solubilize polymers formed 

during oxidation. If this relationship holds true for most biodiesel fuels, it has direct 

effect on the selection and use of this test in a biodiesel specification. It means that the 

results of this test can probably not be used to predict insolubles or deposit-forming 

tendencies at some specific time in the future. Solubility, especially in a biodiesel blend, 

will have a large effect. This test does not measure an induction period or “antioxidation20
capacity” of any type. It does, however, provide a measure of the insoluble/polymer 

forming characteristics of the B100. At this time, though, there is no correlation between 

these test results and insoluble/polymer concentrations at any given time in the future. 

Table 10. Modified D2274 – Polymer Solubility

Tot. Insols, mg/dl Iso-octane Insols, mg/dl 

Test Temperature, °C 95 110 95 110

Soy 7.7 8 2.1 19

Yellow Grease 4.1 2.4 1.4 48 

If a more detailed time/temperature matrix study is conducted, including the solubility 

factor, it should be possible to select a time and temperature suitable for this application. 

Since some previous work has shown that temperatures above 80°C can mask additive 

effects, the study should include B100 samples both with and without antioxidants. 

4.3 ASTM D6468 

The results are presented in Table 11. These results support the widely held assertion that 

the thermal stability of B100, as measured by this test, is very good. Figure 9 presents 

similar results obtained in an earlier program. In that project, both B100 and petroleum 

diesel samples were tested. The B100 samples had very little thermal degradation as 

measured either by reflectance or filter weight. The petroleum diesel samples, however,

showed significant differences in their thermal stability characteristics. 

Table 11. Results for D6468 Tests 

Sample 90 minutes 180 minutes 90 minutes 180 minutes

% Reflectance Gravimetric, mg/100 mL 

Soy 96 96 0.8 1.2

Yellow Grease 96 96 0.4 0.7

21
% Reflectance

% Reflectance

0 20 40 60 80 100 120

Y Data

90 Minutes

180 Minutes

360 Minutes

Gravimetric

Filterable Insolubles, mg / dL

50 40 30 20 10 0 -10

1-B

2-B

3-B

1-P

2-P

3-P

Figure 9. Thermal Stability D6468, Test Results for B100 and Petroleum Diesel Samples 

(See Reference 9). 

4.4 EN 14112 (Rancimat) 

There are two new European standards for biodiesel: EN 14213, for home heating fuel 

and EN 14214 for automotive diesel fuel. Both of these standards contain stability 

requirements based on the Rancimat test. 

• Heating fuels - Fatty acid methyl esters (FAME) - Stability Requirement: Rancimat 

Induction Period @ 110°C ≥ 4 hours. 

• Automotive fuels - Fatty acid methyl esters (FAME) for diesel engines - Stability 

Requirement: Rancimat Induction Period @ 110°C ≥ 6 hours. 

Because the Rancimat test is included in these two European specifications, it is in much 

wider use in Europe as compared to the United States. Also, the amount of data from

running the test on biodiesel samples is greater in Europe. 

22
One significant difference regarding the use of the Rancimat test in Europe vs. the U.S. is 

that most of the B100 produced in Europe is rapeseed-based. Most of the biodiesel 

produced in the U.S. is made from soybeans or yellow grease. Because of its composition 

(lower polyunsaturated content), the rapeseed methyl ester (RME) tends to have 

significantly longer Rancimat induction periods than either soy or yellow grease. Typical 

induction periods for RME are 4-6 hours as compared to 1-2 hours for soy and yellow 

grease. The Rancimat induction periods for the test fuels, at several temperatures, are 

presented in Table 12. 

These much shorter induction periods mean that the sensitivity of the Rancimat is greatly 

reduced when testing typical U.S. B100. Two possible ways to increase the sensitivity 

when testing U.S. B100 are: 

• Treat the B100 with antioxidant to increase the measured induction period. 

However, it is not yet certain that additive effects are linear with different fuels. 

• Run the test at a temperature below the 110°C temperature currently used in the 

European specifications and proposed for U.S. specifications. This approach will 

only work if the oxidation reactions of the methyl esters, are unchanged by 

temperature changes in the range suggested. 

Table 12. Rancimat Test Results 

Sample 

Temperature, °C Induction Period, h 

Soy 120 0.45, 0.48, 0.50, 0.51 

Yellow Grease 120 2.19, 2.30, 2.31, 2.40 

Soy 110 0.67, 0.61, 0.77, 0.65 

Yellow Grease 110 4.81, 5.02, 5.11, 4.80 

Soy 100 0.88, 0.89

Yellow Grease 100 10.01, 10.24 

Soy 130 0.43, 0.42

Yellow Grease 130 1.07, 0.87 

Soy/Yellow Grease Blends 

25/75 

50/50 

75/25 

110

1.63, 1.42 

1.18, 1.23 

0.94, 0.92 

Figure 10 is an Arrhenius Plot of the Rancimat results for several samples of B100. Note 

the significant changes in induction period, for some of the samples, as the temperature 

decreases. This plot suggests that, for the 100°C to 130°C range, it should be possible to 

change the test temperature of the Rancimat without significantly changing the reaction 

mechanisms. 

23
Arrhenius Plot of Rancimat Results

1000/T (K)

2.45 2.50 2.55 2.60 2.65 2.70

h

0.01

0.1

1

10

100

European Specification,

Figure 10. Arrhenius Plot of Rancimat Induction Periods at Selected Temperatures

4.5 ASTM D3241 (JFTOT) 

The JFTOT results are presented in Table 11 and plotted in Figure 11. The soy sample 

shows a steady increase in deposit formation as temperature rises. It is unknown whether 

deposit volume would reach a maximum at some higher temperature. The yellow grease

appears to have reached a maximum at some temperature below 225°C and is decreasing 

slightly between 225°C and 230°C, at which temperature the deposit volume appears to 

remain constant with increasing temperature. 

Because of the small sample size, the data are inconclusive. Further study to evaluate this 

test method is warranted. The JFTOT could be a significant tool for studying high 

temperature deposit formation with B100. However, the JFTOT is not likely to be useful 

as an oxidation stability test for specification purposes. Multiple tests would be required 

to establish the induction period of a given B100; the time and cost of which could be 

prohibitive. 

24
Figure 11. JFTOT Tube-Deposit Volume versus Temperature 

Test Temperature, oC

220 225 230 235 240 245 250

Deposit Volume, cm3

2.0e-6

4.0e-6

6.0e-6

8.0e-6

1.0e-5

1.2e-5

1.4e-5

Soy

Yellow Grease

Table 13. Results of Testing B100 Samples by ASTM D3241 (JFTOT) 

Sample Test Temperature, °C Deposit Rating Deposit Volume±

, cm3

Soy 225 1A* 8.75 x 10-6

235 1A 1.10 x 10-5

245

1A 

1.31 x 10-5

Yellow Grease 225 2 1.06 x 10-5

230 2 4.22 x 10-6

235 2 4.16 x 10-6

*A= Abnormal deposit as compared to typical petroleum jet fuel deposits. 

± Deposit volume was measured with an ellipsometric, tube-deposit, measuring instrument. 

4.6 HPDSC 

The biodiesels were analyzed using a modified version of ASTM D5483, rampedtemperature method. The results obtained are presented in Table 14. The analyses 

indicate that this test does not give useful results regarding the oxidation stability of 

B100. There is insufficient differentiation between the samples under the conditions used. 

The heating started at room temperature and increased at 5°C/min up to 200°C. 

25
Table 14. HPDSC Results for Biodiesel Plus Antioxidants (Temperature 

Ramp = 10°C/min) 

Biodiesel Additive, 500 ppm 

(See Table 11 for Additives) 

Oxidation Induction Time, 

OIT, min 

None 15.24

04-202 14.66

04-203 14.48

04-204 14.77

Soy

04-205 14.93

None 14.92

04-202 15.70

04-203 15.35

04-204 15.23

Yellow

Grease 

04-205 15.34

4.7 IP Method 306 (Modified) 

The results for this testing, given in Table 15, are similar to those for the modified 

D2274. They again demonstrate the effect of solubility on the measured results, with 

relatively high levels of iso-octane insolubles because of the relatively high testtemperature of 120°C. This method could be used in lieu of D2274 although it is not as 

familiar in the U.S. diesel fuel community as is D2274. Also, the work-up procedures for 

this test are slightly more involved than D2274. 

Table 15. Results for Testing of B100 by Modified IP 306 

Biodiesel Total Insolubles,

mg/L (Filt/Adh) 

Soluble Acidity

(mg KOH/g) 

Volatile 

Acidity

(mg KOH/g) 

iso-octane Insoluble 

Material (mg)

Soy 15.6 / 8.4 2.5 2.3 30 

Yellow 

Grease 

11.2 / 8.9 2.0 1.8 135 

4.8 Antioxidant Additives 

Because of the questions regarding test temperatures and additive response with the 

D2274 test (See Section 4.2 above), the decision was made to use the Rancimat method 

for the antioxidant efficacy study. In addition to the food grade antioxidants traditionally 

used in B100 (such as BHT and TBHQ) we obtained 4 petroleum antioxidants from a 

petroleum additive supplier for evaluation. The additives are described in Table 16. The 

Rancimat test results are presented in Tables 17 and 18. Of the food grade additives, only 

TBHQ caused a significant increase in the induction time for the soy B100. The food 

grade additives gave a slight improvement in the induction times of the yellow grease. 

The petroleum additives caused a significant increase in the induction period of the 

yellow grease. Selected test results are also plotted in Figures 12 and 13. Figure 12 

demonstrates that additive effects tend to be linear but not equivalent. This relationship 

could allow biodiesel suppliers to determine necessary treat rates, with a given additive, 

to reach a pre-selected Rancimat induction period. That is especially useful if the 

26
Rancimat test is selected for D6751 specification purposes. These results demonstrate 

that the Rancimat can discriminate antioxidant additives. However, it is possible that a 

lower test temperature, say 90°C or 100°C will be required when evaluating the effects in 

some biodiesel samples. 

Additive Concentration, ppm

0 200 400 600 800 1000 1200

Ranci

mat Induction Period, h

0.0

0.5

1.0

1.5

2.0

2.5

Untreated Soy

TBHQ

Tenox 21

BHT

Figure 12. Antioxidant Efficacy for Three Food-Grade Additives via Rancimat at 110°C, soy

based biodiesel 

Additive

 202 202-A 203 203-A 204 204-A 205 TBHQ Tenox 21 BHT

Rancimat Induction Period, h 0.0

0.5

1.0

1.5

2.0

2.5

Untreated Soy

Figure 13. Comparison of Antioxidant Effects for Selected Petroleum and Food-Grade 

Additives, Rancimat at 110°C. 

27
Table 16. Description of Additives Used 

Additive Description

BHT Butylated phenol

TBHQ Hindered phenol

Tenox 21 20% TBHQ+1% citric acid

04-202 Substituted phenol liquid 

04-203 Butylated phenol

04-204 Alkylated phenol, functionalized 

04-205 Mixture phenol/amine types 

Table 17. Effect of Food-Grade Antioxidants -- Rancimat Testing 

Sample Test Temperature Induction Period, h 

Soy + TBHQ 110 10 ppm: 0.83,0.70 

50 ppm: 0.75, 0.78 

100 ppm: 0.95, 0.96 

500 ppm: 1.98, 1.82 

Soy + Tenox 21 110 10 ppm: 0.67, 0.70 

50 ppm: 0.72, 0.67 

100 ppm: 0.65, 0.67 

500 ppm: 0.76, 0.79 

1000 ppm: 0.83, 0.90 

Soy + BHT 110 10 ppm: 0.76, 0.75 

50 ppm: 0.65, 0.68 

100 ppm: 0.75, 0.73 

500 ppm: 1.00, 0.88 

Table 18. Effect of Petroleum Antioxidants -- Rancimat Testing 

B100 Additive Induction Period, h @ 500 ppm 

110°C 120°C 

04-202 0.92, 1.02 0.45

04-203 2.03, 2.08 0.99, 1.23 

04-204 2.26, 1.67 0.83, 0.87 

Soy (04-200) 

04-205 0.77, 0.80 0.43, 0.41 

04-202 16.12, 14.47 3.86, 5.83 

04-203 12.65, 12.33 5.11, 5.48 

04-204 24.56, 25.14 7.77, 8.71 

Yellow Grease 

(04-201) 

04-205 17.07, 16.82 7.86, 7.65 

4.9 Oxidation Stability of Commercially Available B100 Fuels 

The Rancimat results presented in Tables 19 and 20 are for B100 samples taken from

various sources. The samples in Table 19 were obtained from a survey of commercially 

available B100 throughout the United States. The samples in Table 20 are a mix of 

samples on-hand at SwRI during this project. More complete descriptions of any of these 

samples were not readily available. Taken all together, these results demonstrate the 

varied range of results typical of current U.S. biodiesel. The most significant point is that 

28
nearly all of these samples have induction periods well below the European specification 

of 6 hours, minimum. 

Table 19. Rancimat Test Results for B100 Fuel Quality Survey Samples 

Induction Period, h 

Lab ID 

(No Description was 

Available) 110°C 120°C 

04-132 0.66, 0.68 0.49, 0.50 

04-133 0.55, 0.39 0.14, 0.18 

04-135 1.64, 1.57 0.74, 0.86 

04-136 0.52, 0.51 0.39, 0.40 

04-137 1.21, 1.24 0.54, 0.60 

Table 20. Rancimat Results for Samples of Commercially Available B100 Fuels 

from Various Sources 

Sample Description Rancimat Induction Period, h 

CL04-395, Soy B100 plus antioxidant 5.12, 5.05 

CL04-396, Y.G., 2763-001C 5.37, 5.32 

CL04-397, Soy B100 0.18, 0.55 

CL04-398, Canola B100 4.86, 4.81 

B 100 0.9 

B 100 + antioxidant 1.3 

Rapeseed Methyl Ester 1.5 

Rapeseed Methyl Ester plus 100 ppm BHT 4.9 

4.10 Tests of B20 by D2274 and Rancimat 

B20 blends with low sulfur diesel and kerosene were prepared using both the soy and 

yellow grease biodiesel samples. The modified D2274 and Rancimat results are presented 

in Table 21. This limited set of data suggests a correlation between the results of these 

two tests for B20 blends. Further investigation is warranted. As ultra-low sulfur diesel 

fuel becomes more available on the open market, there should be some specific studies of 

B20 blends with these fuels. 

Table 21. Rancimat Test (110°C) Results for B20 Blends 

Sample Modified D2274, mg/100mL 

Filt/Adh/Total

Rancimat Induction 

Period, h 

CL04-221, Soy / Low Sulfur 

Diesel 

3.1/0.3/3.4 

2.7/0.3/3.0 

11.23 

CL04-222, YG / Low Sulfur

Diesel 

0.0/0.1/0.1 

0.0/0.1/0.1 

> 24 

CL04-223, Soy / Kerosene Fuel 0.0/0.2/0.2 

0.0/0.2/0.2 

> 24 

CL04-224, YG / Kerosene Fuel 0.0/0.1/0.1 

0.0/0.1/0.1 

> 24 

29
5.0 Conclusions on Selection of an Oxidation Stability 

Test Method for B100 

5.1 Test Methods 

The test methods evaluated during this project were: 

• ASTM D4625, Standard Test Method for Middle Distillate Fuel Storage Stability 

at 43°C (110°F). 

• ASTM D6468, Standard Test Method for High Temperature Stability of Distillate 

Fuels. Modified for gravimetric determination of insoluble formation. 

• ASTM D2274, Standard Test Method for Oxidation Stability of Distillate Fuel Oil 

(Accelerated Method). Modified, specifically for use with biodiesel, as described 

in this report. 

• ASTM D3241, Standard Test Method for Thermal Oxidation Stability of Aviation 

Turbine Fuels (JFTOT Procedure). 

• EN 14112, the Rancimat Test, Fat and oil derivatives. Fatty Acid Methyl Ester 

(FAME). Determination Of Oxidation Stability (Accelerated Oxidation Test). 

• Modified IP 306, Determination Of Oxidation Stability Of Straight Mineral Oil. 

• ASTM D5483, Standard Test Method for Oxidation Induction Time of 

Lubricating Greases by Pressure Differential Scanning Calorimetry. 

The test methods were evaluated for their applicability to the measurement of biodiesel, 

ability to discern additive effects, and ability to discriminate between biodiesel samples 

of various levels of oxidation stability. 

ASTM D4625 is an excellent method for estimating the long-term storage stability of 

middle distillate petroleum fuels. One week of storage at 43°C is widely accepted as 

equivalent to 4 weeks at 15°C (underground, ambient storage). While the same

relationship has yet to be proven for B100, most researchers have tended to accept that 

the correlation holds. This makes D4625 an excellent research method but it is not 

acceptable as a specification test. 

ASTM D6468 has existed in nearly the same form (albeit different names) for over 60 

years. Its very short 90-minute test time makes it a very attractive test for quality 

assurance and quality control. The 150°C test temperature makes this test quite severe. 

There is no active addition of air or oxygen to the fuel during testing so this test is not as 

useful for measuring oxidation stability. Also, this test method has historically relied on 

estimating the amount of insolubles formed based on the darkness of the material trapped 

on a filter pad. Biodiesel insolubles tend to be far less dark in color than petroleum diesel; 

and, as such, are more difficult to quantify using optical methods. Gravimetric 

measurement of insolubles provides more reliable quantification. Biodiesel tends to be 

very thermally stable but less oxidatively stable when compared to petroleum diesel. This 

test method does not provide a useful discrimination between biodiesel fuels of varying 

quality. This test may, however, be useful for B20 but more work is needed. 

30
ASTM D3241 (JFTOT) will require additional study to determine if it can be used to 

measure the oxidation stability of biodiesel. This test is quick and simple to perform.

There are numerous methods for quantifying the deposits formed although most are 

visual so not as useful. The ellipsometric tube-rating instrument shows promise since 

deposit color does not affect the measurement. This test method is a specification test for 

aviation fuel so there is already acceptance of the results for specification purposes. The 

greatest strength of this test may be as a measure of the tendency of a biodiesel to form 

deposits on a hot metal surface. In general, there is currently insufficient data to 

recommend this test as a specification test but it deserves additional study. 

IP 306 is a strong candidate for a specification test. It is oxidizing and it measures both 

total and iso-octane insoluble material. It uses a relatively small 100-mL sample size, 

which tends to reduce the precision and sensitivity of the method. Also, it is not as widely 

used in diesel fuel test laboratories so the glassware is not as commonly found. This test 

method could be used in a specification but is so similar to the more commonly run 

D2274 that D2274 is probably a better choice. 

ASTM D5483 as used in this study, failed to give useful results regarding the relative 

stabilities of various fuels. Still, research into the use of DSC continues and it is 

reasonable to assume that a suitable method can be developed. At such time, the method 

should be considered a viable candidate for inclusion in a specification. 

At this time, there are two likely choices for an oxidation stability test for B100 (D6751). 

Those two choices are the Rancimat test and some modification of ASTM D2274. 

5.1.1 Rancimat Test 

This test is well established in Europe and is included in several European specifications. 

It is quick and simple to run and could be completed in a single work shift (a plus for

using it as a specification requirement). It provides a repeatable measure of the 

antioxidant capacity of the biodiesel although the relationship of that measure to the field 

has not been established. 

This test does not give any measure of polymer forming tendencies. It is believed that 

some types of B100 are more prone to forming polymers (and insolubles) than others. 

This could be a factor when some formation of insolubles is considered acceptable. 

5.1.2 Modified D2274 

Unlike the Rancimat, this test gives no measure of induction period. It is presumed that 

all antioxidant capacity in the B100 is consumed during the test. Hence, this test is more a 

measure of the tendency of the B100 to form polymers and insolubles. For those 

concerned about the formation of these materials, D2274 is probably a better screening 

tool/test. 

31
The current versions of the test are at least 16 hours long but it may be possible to shorten 

the aging period to allow completion within a single shift. Doing so would require 

substantial additional work. 

It may also be necessary to change the test temperatures of both of these tests to allow 

better discrimination between samples and also additives. 

5.2 Specification Limits 

The European specification for B100 includes a 6-hour minimum Rancimat induction 

period. The vast majority of U.S. biodiesels have Rancimat induction periods that range 

between 1 and 4 hours. Limited testing has shown that excessive amounts of currently 

used antioxidants would be required before most U.S. fuels could meet the European 

specification. Additionally, to date there has been no controlled study of the minimum 

induction period required to minimize the chance of problems in the field. Many in the 

industry point to the lack of reported problems, linked to unstable biodiesel, as proof that 

U.S. fuels are for the most part acceptably stable. As such, it is expected that any ASTM 

specification that uses the Rancimat will probably have a lower minimum induction 

period, perhaps 4 hours rather than 6. 

In contrast, biodiesel tends to form more insolubles in tests like D2274 when compared to 

petroleum diesel. Historically, specifications that include D2274 have set limits of 1.5 to 

2.5 mg/100mL, maximum. This would likely be too restrictive for U.S. biodiesel (and 

perhaps European fuels as well). A slightly relaxed requirement of perhaps 4.0 mg/100, 

maximum, may be more appropriate. However, inclusion of iso-octane insolubles 

measurement will alter that limit. Additional work is needed to find an acceptable limit 

with this test method. 

5.3 Antioxidants 

In general, each of the test methods showed the relative efficacy of various antioxidants. 

The relative ranking was similar but not always exactly the same. Test times and 

temperatures seem to have a significant effect with some of the tests, such as D2274. 

 

Historically, food-grade antioxidants have been the additives of choice for biodiesel. This 

project was able to show that petroleum antioxidants can give equal or greater protection 

against oxidation. Antioxidant efficacy in biodiesel blends, especially with ultra-low 

sulfur diesel, must be studied further. It is assumed that ULSD will be far more 

susceptible to oxidation since natural inhibitors are removed along with the sulfur 

compounds. Some combination of food-grade and petroleum additives may be required in 

the near future. 

32
List of Acronyms 

ASTM American Society of Testing and Materials, ASTM International, a 

standards setting organization 

B100 100% Biodiesel 

B20 A blend of 20% biodiesel in petroleum diesel 

BHT Butylated hydroxy toluene 

DSC Differential scanning calorimetry 

EN European Normalization

FAME Fatty acid methyl ester 

GCMS Gas chromatography mass spectrometry 

HPDSC High pressure differential scanning calorimetry 

IP Institute of Petroleum

JFTOT Jet fuel thermal oxidation test 

NREL National Renewable Energy Laboratory 

RME Rapeseed methyl ester 

TBHQ Tert-butyl hydroquinone 

ULSD Ultralow sulfur diesel (15 ppm S in the US). 

USDOE United States Department of Energy 

33
References 

 

1

 All ASTM Methods Published by ASTM International, West Conshohocken, PA; 

www.astm.org 

2

 Stavinoha, L.L. and Howell, S. “Potential Analytical Methods for Stability Testing of 

Biodiesel and Biodiesel Blends” SAE Technical Paper No. 1999-01-3520 (1999). 

3

 McCormick, R.L. personal communication, 2005. 

4

 FAME Analyses conducted and provided by NREL, Golden, CO. 

5

 Bondioli, P, Gasparoli, A., Della Bella, L., Tagliabue, S. "Evaluation of Biodiesel 

Storage Stability Using Reference Methods” Eur. J. Lipid Sci. Technol. 104 777-784 

(2002). 

6

 de Man, J.M., Tie, F., and de Man, L. “Formation of Short Chain Volatile Organic 

Acids in the Automated AOM Method: J. Am. Oil Chem. Soc. 64 993-996 (1987). 

7

 Miyata, I., Takei, Y., Tsurutani, K., and Okada, M. “Effects of Bio-Fuels on Vehicle 

Performance: Degradation Mechanism Analysis of Bio-Fuels” SAE Technical Paper No. 

2004-01-3031 (2004). 

8

 “Determination of Oxidation Stability of Straight Mineral Oil,” [Formerly Institute of 

Petroleum, now Energy Institute] Standard Test Method 306.

9

 Westbrook, S.R. and Stavinoha, L.L., ‘Biodiesel And B20 Blends: Stability Test 

Methods And Stability Characteristics,” IASH 2003, the 8th International Conference on 

Stability and Handling of Liquid Fuels, Steamboat Springs, Colorado, September 14-19, 

2003. 

10 Fang, H. L., “Deposit Formation of Biodiesel,” Presentation, August 2004. 

34
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4. TITLE AND SUBTITLE

An Evaluation and Comparison of Test Methods to Measure the 

Oxidation Stability of Neat Biodiesel 

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NREL/SR-540-38983 

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Southwest Research Institute 

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14. ABSTRACT (Maximum 200 Words)

The purpose of this project was to compare and evaluate several candidate test methods for evaluating oxidation 

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