Determining the Influence of Contaminants on
Biodiesel Properties
Final report prepared for:The Iowa Soybean Promotion Board

Submitted by:
Jon H. Van Gerpen
Earl G. Hammond
Lawrence A. Johnson
Stephen J. Marley
Liangping Yu
Inmok Lee
Abdul Monyem

Iowa State University

July 31, 1996

Table of Contents

1. Introduction
2. Background
2.1 ASTM Specifications
2.2 Nationalfor Diesel Fuel Oils (D975)

Biodiesel Board Specifications

for Biodiesel

3. Results
3.1 Unsaponifiable

matter

3.1.1 Crystallization

Onset Temperature

3.1.2 Cetane Number
3.2 Water
3.2.1 Water Solubility

8

3.2.2 Microorganisms

10

3.3 Free Glycerin

11

3.4 Bound glycerin

12

3.4.1 Crystallization
Diglyceridesof Pure Monoglycerides

and14

3.4.2 Monoglyceride and Diglycerides Formed
During Incomplete Transesterification

16

3.4.3 The Effect of DG/MG

17

Ratio on Crystallization

3.4.4 The effect of bound glycerin on the viscosityof biodiesel and diesel fuel3.4.5 Discussion of the Bound Glycerin Specification
3.5 Alcohol

18
19
20

3.6 Free Fatty Acids, Soap, and Catalyst

21

3.7 Oxidation

24of Biodiesel

4. Conclusions

28

5. References29

1. Introduction
Hic&ese/, as used in this report, is defined to be the alkyl monoesters of vegetable oils or
animal fats. It is produced by chemically reacting the oil or fat with an alcohol in the presence ofa catalyst. The products of this reaction are the monoesters, known as biodiesel, and glycerin,
which is a high value byproduct. Because of its low cost, methanol is the most common alcohol
used but ethanol is used in some cases. In Europe, rapeseed oil is the most common vegetable oil
feedstock and in the United States, soybean oil is generally used. AI1 of the work presented in thisreport uses biodiesel produced from methanol and soybean oil. This particular form of biodiesel isoften called methyl soyate.
The American Society for Testing and Materials (ASTM) and the American Society ofAgricultural Engineers (AWE) are currently developing specifications for biodiesel. Althoughthese specifications are still in draft form, they will almost certainly contain limits on the percent
esterification, the cloud point, the pour point, the amount of free glycerin and total glycerin, as
well as many other properties. The limits on these properties are set as compromises between thedesire of the engine manufacturer to have no contaminants and the desire of the fuel supplier tohave levels that will be easy and inexpensive to meet. It would be very useful to have some data
to guide the standards setting process Biodiesel standards that are too strict will increase thecost of the fuel and standards that are too lax will increase the likelihood that consumers willencounter fuel-related problems. Both possibilities could impact the market acceptance of
biodiesel. The problem directly impacts biodiesel cost since the lack of knowledge about
contaminant effects will probably result in very conservative standards that will be expensive tosatisfy.
The amount of data on biodiesel properties and its effect on diesel engine performance andemissions has greatly increased over the past three years, due in large part to the financial supportof soybean farmers through their funding of the National Biodiesel Board. A serious problem has
now developed because it has been found that different researchers, running the same
standardized tests on biodiesel, have obtained very different results. For example, until recently,
the cetane number of methyl soyate was believed to be about 48. Now data has been presented
that the cetane number may actually be as high as 60. This change is well beyond measurement
error and is probably attributable to fuel contamination Thus, in addition to the need to control
fuel quality to protect the engine, there is also the need to understand the general relationshipbetween contaminants and fuel properties.
The objective of this project was to develop the database that would provide a basis forsetting realistic specification values for biodiesel. This report will discuss each of the expectedcontaminants in terms of their impact on engine life and performance and present data to justify aparticular specification value. The contaminants to be considered include water, free glycerin,bound glycerin, alcohol, free fatty acids, soaps, and residual catalyst. During the reporting year,
unsaponifiable matter, microorganisms, and oxidation products were added to the list of important
contaminants, and the effects of their presence on the properties of biodiesel were also studied,This project was jointly funded by three entities: the Iowa Soybean Promotion Board, theIowa Energy Center, and the Iowa Department of Agriculture’s Renewable Fuels Committee,
Due to a delay in receiving the funds from the Iowa Department of Agriculture, a portion ofthe
project has not been completed Rather than request an extension of the overall project, we arereporting on the results of the project to date and not including the portion ofthe project yet to be2completed The remaining work consists primarily of engine testing of the impact ofmonoglycerides on injector deposits. A second report will be submitted to all fknding agencies
when this work is complete. We expect this report to be finalized by September 30, 1996.

2. Background
This section presents the specification values for diesel fuel and biodiesel. The American
Society for Testing and Materials maintains the standard for petroleum-based diesel fuels. It
covers three grades of diesel fuel although No. 2-D is the most common grade used in trucks andagricultural equipment. No 1-D is used as a blending agent during the winter to improve diesel
fuel cold flow properties.
2.1 ASTM Specifications

for Diesel Fuel Oils (D975)

ASTM standard D97S covers three grades of diesel fuel oils.
1. Grade No. 1-D: A light distillate fuel for applications requiring a higher volatility fuelfor rapidly fluctuating loads and speeds as in light trucks and buses. The specification for thisgrade of diesel fuel overlaps with kerosene and jet fuel and all three are commonly produced fromthe same base stock. One major use for No. 1-D diesel fuel is to blend with No. 2-D during
winter to provide improved cold flow properties.
2 Grade No. 2-D: A middle distillate fuel for applications that do not require a highvolatility fuel. Typical applications are high speed engines that operate for sustained periods athigh load.
3. Grade No. 4-D: A heavy distillate fuel that is viscous and may require fuel heating forproper atomization of the fuel. It is used primarily in low and medium speed engines.
ASTM D975 specifies the property values shown in Table 1 for these three grades of diesel fuel.

2.2 National Biodiesel Board Specifications

for Biodiesel

The National Biodiesel Board (NBB) maintains a specification for biodiesel that iscurrently under review for acceptance as a standard by the ASTM. The NBB maintains a list of
biodiesel fuel suppliers who have agreed to supply fuel meeting these specifications. The
specification values are shown in Table 2. The values given in the table were adopted based on
European experience with rapeseed methyl esters. It is not known whether the values given areappropriate for biodiesel produced and consumed in the United States. The objective of thisproject is to investigate whether the specifications associated with contaminants are valid.

3
Table 1. ASTM D 975 Specifications

Propertv

for Diesel Fuel

ASTM test

Grade 1-D

Grade 2-D

Grade 4-D

Flash Point (oC)

D93

38

52

55

Water and Sediment (%)

D1796

0.05

0.05

0.50

Distillation Temperature
90% recovered, min
max

D86
none
288

282
338

none
none

Kinematic Viscosity
min
max

D445
1.3
2.4

1.9
4.1

5.5
24.0

Ramsbottom carbon residueon 10% distillation residue

D524

0.15

0.35

none

Ash, % mass, max

D482

0.01

0.01

0.10

Sulfur, % mass, max

D129

0.50

0.50

2.00

Copper Strip corrosion
rating, max 3 hr at 50°C

D130

No. 3

No. 3

none

Cetane Number, min

D613

40

40

30

Cloud point, OC, max

D2500

Guidance only.
Should be 6°C higher than the tenth
percentile minimum ambient temperature
for the region
For Iowa,Month10th % minimum temp
Ott
-2°C
Nov
-13
Dee
-23

Jan

-26

Feb
Mar

-22
-16

4

Table 2. The National Biodiesel Board Specifications

for Biodiesel

Proper&

ASTM Method

Limitsunits

Flash Point

D 93

100 min“C

Water and Sediment

D 17960.050 max

Vol. %

Carbon Residue, 100% sample

D 45300.050 maxwt %Sulfated Ash

D 874

0.020 maxwt %Kinematic Viscosity, 40°C

D 445

1.9 - 6.0mm2/sec

Sulfur

D 26220.05 max

w-t %Cetane Number

D 613

40 minCloud Point

D 2500By customer

Copper Strip Corrosion

D 130

No. 3b max

Acid Number

D 664

0.80 max

mg KOH/g

Free Glycerin

GC

0.020 max

w-t %Total GlycerinGC

0.240 max

wt %

“C

3. Results
This section presents the results of investigation into the correct values of contaminantconcentrations in biodiesel to be used as an engine fuel. The contaminants considered are:unsaponifiable matter, water free glycerin, bound glycerin, alcohol, free fatty acids, soaps, residual
catalyst, and oxidation products.
3.1 Unsaponifiable

matterTypical crude soybean oil contains as much as 1 6% rrnsapon~fiahle matter, which consistsof plant sterols, tocopherols and hydrocarbons as major components, with very small quantities ofpigments and minerals. The unsaponifiable matter is not affected by ester preparation, so it islikely to be present in similar amounts in biodiesel to its level in crude soybean oil. The
compounds in the unsaponifiable matter have varying amounts of unsaturation, and include open

chain, cyclic aliphatic and aromatic functional groups which are similar to many of the compounds
present in diesel fuel The unsaponifiable matter may have no harmful effects on engine
performance except possibly for a change in the crystallization onset temperature caused by polarsterols. Therefore, the effect of the unsaponifiable matter on crystallization properties of neatesters and 20% esters/go% diesel tie1 blends were studied. A second issue related theunsaponifiable matter is whether it has any impact on the cetane number of the biodiesel. Thiswas investigated also.
3.1.1 Crystallization

Onset Temperature

Simulated unsaponifiable matter that resembles that found in crude soybean oil wasformulated with 70% p-Sitosterol, 14% a-tocopherol, 8% Squalane, and 8% Squalene. Distilled
methyl soyate (purchased from Interchem Environmental of Kansas City, MO) was prepared withvarious unsaponifiable levels (O%, l%, 2%, and 3%), and 20% (by volume) blends of methyl
soyate and No. 1 diesel fuel were prepared with the same concentrations of unsaponifiables. Two
separate samples were prepared for each unsaponifiable level, and two measurements were madefor each sample. Unsaponifiable matter was measured by the AOCS Official Method Ca 6a-40.
Crystallization properties were characterized by the cloud point (ASTM Standard Method
D2500), pour point (ASTM Standard Method D97) and the crystallization onset temperature(Tco) determined by differential scanning calorimeter (DSC) analysis. The Tco was determinedas the end point (where the tangent of the peak meets the baseline) of the highest melting
transition peak, that is, the lowest temperature at which the sample was completely liquid.
The content of unsaponifiable matter in various commercial and experimental methyl
soyates and the soybean oils from which they are derived were determined and are shown in Table
3. Replications agreed well, and the Interchem methyl soyate, which was double-distilled,contained practically no unsaponifiable matter. When 1% of the simulated unsaponifiable matter
was added to the Interchem methyl soyate, the sample was analyzed as having 0.91%
Table 3. Unsaponifiable

matter content of various methyl esters and soybean oils
Unsaponifiable Matter (%)
Samples
Rep. 1
Rep. 2Average
Methyl esters
0.06
0.00
0.03
(purchased from Interchem)
Methyl esters (Interchem)
0.91
0.90
0.91
with 1% unsanonifiables added
Methyl esters
0.50
0.48
0.49

(Purchasedfrom Calgene)
Crude soybean oil (West Central Coop.)
Methyl esters made from West Central soybean
oilCommercial RBD soybean oil
Methyl esters made from commercial RBD
sovbean oil

0.41
0.49

0.46
0.51

0.44
0 50

0.48
0.44

0.43
0.52

0 46
0.48

6

Table 4. The effect of unsaponifiable matter on crystallization
esters and 20% esters-kerosene blends

properties of neat methyl

Values \vlth diffcrcnt superscriptm the samerow are significantly different at ~0.05

unsaponifiable matter, which indicated that 9 I % of the added unsaponifiable matters wererecovered by the extraction procedure of the analysis method. The amounts of unsaponifiable
matter measured in several samples of soybean oil and the methyl esters produced from thosesamples were all between 0.4 to 0.5%, which is on the low end of the expected range. The
amounts of unsaponifiable matter in both crude and RBD oils, were not significantly changed (ata=0 05) during the preparation, transesterification, and washing of the methyl soyates.
The effect of unsaponifiable matter on the crystallization properties of biodiesel fuels isshown in Table 4. Unsaponifiable matter up to 2% caused no adverse effect on Tco in the neatesters The Tco of ester samples containing small amounts (<2%) of unsaponifiables was evenslightly less than the neat esters because of a melting point depression effect of such “impurities.”
At the 3% unsaponifiable matter level in neat esters, the Tco was significantly increased; however,in the 20% esters/No. 1 diesel fuel blends, there was no significant effect on Tco up to 3%
unsaponifiable matter. Results of cloud and pour point measurements agreed with those fromDSC Only the sample with 3% unsaponifiables had a higher cloud and pour points than theothers w-hen measured in neat esters, and this sample showed no difference in 20% blends withdiesel fuel. It appears that at normally expected levels of unsaponifiable matter, there should be
no deleterious effect on the cold flow properties.
3.1.2 Cetane Number
One major difference between different samples of biodiesel may be whether the fuelcontained the unsaponifiable matter that was originally present in the soybean oil. Most of thesoybean-basedbiodiesel used during 1993 and 1994 in the United States was processed using a
distillation process that removed the unsaponifiable matter along with the glycerin and all othermaterials with higher boiling temperatures than the esters themselves. This fuel, commonly
identified as “double distilled fkel,” may have different properties than undistilled fuel due to theremoval of the unsaponifiable matter.
To investigate the effect of unsaponifiable matter on the cetane number, the same synthetic
unsaponifiable material described above was blended with #2 diesel tie1 at 1% and 2% levels (by7Table 5. Effect of Unsaponifiable Matter on Cetane Number
Cetane Number (ASTM D6 13)
Fuel
45.9
Diesel Fuel
44.2
1% Unsap in Diesel Fuel
2% Unsap in Diesel Fuel
45.6
Methvl Sovate
52 7
1%
Unsap in Methyl Soyate 53.7
3% Unsap in Methyl Soyate 53 4
3% Unsap in Methyl Soyate 52 8

weight) It was not possible to keep more than 2% of this material in solution with diesel fbel.
The unsaponifiable matter was blended with methyl soyate at l%, 2%, and 3% levels.
Table 5 shows the effect of unsaponifiable matter on the cetane number of diesel tie1 andmethyl soyate The data show that the presence of unsaponifiable matter in the fuel has no effecton the cetane number of the fUe1, either for diesel fuel or for methyl soyate. The slight changes incetane number are generally less than the 0.7-O 8 ASTM repeatability specification for cetane
number and are always less than the 2.6-3.1 reproducibility specification.
The currently proposed standards for biodiesel do not contain a specification forunsaponifiable matter. The data presented here do not indicate that such a specification isnecessary.

3.2 Water
Water is a major source of fuel contamination. While fuel leaving a production facility
may be virtually water-free, once it enters the existing distribution and storage network, it will
come into contact with water. Water typically enters fuel tanks through vents and seals as
humidity in the air. The water condenses or is dissolved into the fUe1. Virtually all diesel fuelstorage tanks can be assumed to contain some water. In some cases, water is even used for tankcleaning and as ballast [ 11.
Water in the fuel generally causes two problems. First, it can cause corrosion of engine
fuel system components. The most direct form of corrosion is rust, but water can become acidic
with time and the resulting acid corrosion can attack fuel storage tanks.
The other major problem associated with water contamination is that it contributes tomicrobial growth. There are species of yeast, tingi, and bacteria that will grow at the interface
between the fire1 and any free water that has collected at the bottom of a storage tank. The
organisms produce sludges and slimes that can cause filter plugging [ 11. Some of the organisms
can convert the sulfur in the Abel to sulfi~ric acid which can corrode metal fuel tanks.
Water w-ill be present in the fuel in two basic forms. It will be dissolved in the fuel or as
free water collected at the bottom of the sample. The amount of water dissolved in the fueldepends on its solubility. The amount of free water in the sample depends primarily on the qualityof the “housekeeping” practices followed by the fuel distributors and consumers. Frequently
draining the water from storage tanks, ensuring that vents and seals do not allow rainwater to8enter, and not drawing from the bottom of the tank, should prevent large amounts of free waterfrom entering the system.
To investigate the problem of water contamination, the solubility of water in methyl esters
was measured and compared with diesel fuel. Then, since the primary problem associated withfree water is microbial growth, the tendency of the methyl esters to support microbial growth wasinvestigated
3.2.1

Water

Solubility

The objectives of our study were to compare the solubility of water in methyl esters, 20%esters/80% No. 2 diesel fuel blends, and No. 2 diesel fuel, and to determine the effect of oxidationlevel on the solubility of water in methyl esters. Fuel oxidation was included as a variable since
the polar nature of the hydroperoxides formed during oxidation might cause the oxidized fuel tohave different solubility properties than the unoxidized fuel.
Figure 1 shows the solubility of water in diesel fuel. This plot was constructed from afigure that used different units (ml/gal) in reference [2]. At 2S’C, the solubility of water in diesel
fuel is only about 60 ppm. This is quite low and probably results in no adverse effects. ASTM
standard D975 for No. 2 diesel fuel allows up to 0.05% water (500 ppm) which includes both
dissolved and free water. With No. 2 diesel tie], the specification assumes that most of the water

2040
Temperature,

60
deg C

Figure 1. Solubility of water in diesel fuel. (Based on [2])80

9is present as free water. The tests conducted as part of this study compared the solubility ofwater in methyl esters with its solubility in diesel fuel
The fuels (highly-oxidized Interchem methyl esters with a peroxide value (PV) =124,
freshly made methyl esters with a PV=20, 20% blend of methyl ester in 80% No. 2 diesel fuel, andNo. 2 diesel mel) were vigorously mixed with an equal volume of water. The water content in thefuels was then determined by Karl Fischer titration after 0, 1, 3, 18, 24, and 120 hours of mixing.
Table 6 summarizes the results from the water solubility tests The data shown for zero
hours was the baseline case before water was added. Karl Fischer (K-F) titration values were
zero or negative when samples of highly-oxidized Interchem methyl soyate were titrated This
result may have been caused by re-oxidation of the iodine ion, formed during blank titration of thesolvent methanol, by the peroxides in the highly oxidized methyl soyate (PV=124). Slight signs of
the re-oxidation were observed during the titration of freshly-prepared methyl soyate (PV=20),
however. positive titrations were obtained with this sample. The freshly-prepared esters, vacuumdried after washing with water, had a fairly low water content of about 40 ppm, which was almost
as low as that of No. 2 diesel fuel When mixed vigorously with water, the methyl soyate wassaturated with water in one hour, and the water level in the esters did not increase after 5 days of
mixing. There was no change in the peroxide value of the esters samples during the mixing. The
saturation level of water in methyl soyate was about 40 times greater than that of diesel fuel andwas close to the 1500 ppm level, which was observed for water-washed esters before vacuum
drying. The diesel fuel had a very low water content at the start of the test, about 28 ppm, and noincrease was observed after 18 hr of vigorous mixing The differences between the values
reported for the solubility, 28 ppm, 8 1 ppm, and 30 ppm, are not considered to be statisticallysignificant. Their average is close to the value reported in Figure 1. The 20% blend of methyl
soyate in 80% diesel fuel had a water content similar to diesel fuel, and there was no increase inwater content after an extended period of vigorous mixing. These results suggest the possible
separation of free water when water-saturated esters are blended with diesel firel.
Table 6. Water contents (ppm) in various methyl
diesel fuel blend after vigorous mixing with water

OHEt
Methyl soyate(PV=l24, Interchem)
Methyl soyate(PV=20)20% Blend(PV=20)
Diesel fuel(No.2)

1HR

soyates, No. 2 diesel fuel and 20% estersfor various periods at 25°C.”

3HR

18HR

1D

5D

0

0

0

37’

1460”

1595”

1255”

1225b

40”

37”

38”

33”

45”

28”

81”

0

30”

*The values are the averageof duplicate measurements.
Values nlth different superscript in the same row are significantly different at ~0.05

10
3.2.2 Microorganisms
One advantage of biodiesel, over petroleum-based diesel tiel, is its biodegradability,
however, this biodegradability may allow biodiesel to be more susceptible to microbial
contamination than petroleum fuels. Our study was focused on comparing the bioavailibility of
biodiesel to diesel fuel for different types of microorganisms.
Dirt samples were taken Corn a methyl soyate storage site and were inoculated into aliquid media containing various nitrogen sources (Yeast N-base and Pseudomonas basal mineral
N-base) and carbon sources (No. 2 diesel fuel and methyl soyate fuels) and the microbial growth
was compared The culture media were incubated on a shaker in the dark at room temperature,and their growth was monitored by measuring the optical density of the aqueous phase of theculture media with a spectrophotometer. To isolate the microorganisms from the various
cultures, aliquots of the culture media were spread on agar plates (Yeast nitrogen base with 0.5%
glucose and Pseudomonas basal mineral medium with I .4% glucose) and incubated at room
temperature. The single colonies were &t-ther streaked on additional agar plates until purecultures were obtained. The morphology of the isolated microorganisms was observed.
The growth of microorganisms on the various liquid media are shown in Table 7. The
yeast N-base with 1% methyl ester media had no significant increase in turbidity until 6 days
incubation, but at 6 days, visible mold was found in the liquid medium although the solution was
still clear. Significant growth was found after 3 days incubation in the yeast N-base with 1%
diesel fuel. Pseudomonas N-base with 1% methyl ester showed growth after 2 days incubation,
while Pseudomonas N-base with 1% diesel fuel showed growth afler 3 days incubation. The same
trend of growth was also found on the agar plates. However, cultures from diesel and methyl
ester containing liquid media indicated different types of microbial growth on the agar plate. Twocultures with different morphologies were isolated from the yeast N-base diesel medium on theyeast N-base agar plates. Four cultures were isolated Corn the yeast N-base methyl ester medium
on the yeast N-base agar plates Of these, two had the same morphology as those isolated fromthe diesel fuel medium and one of the remaining isolates was mold. On Pseudomonas N-base agar
plates, two cultures were isolated from the Pseudomonas N-base diesel fuel medium and four
from the Pseudomonas N-base methyl ester medium. Of these, one appeared to be the same asthat isolated from the diesel fuel medium and two were molds. No molds were observed or
isolated from any of the media with diesel fuel as the carbon source, therefore, at this point, it
seems that methyl ester is more hospitable to molds than diesel fuel. Further studies are needed to
identify the isolated cultures, and to find the storage conditions for biodiesel that will prevent
microbial growth.
Table 7. The growth of microorganisms

on various diesel and methyl soyate liquid media’Absorptance
2 days3 days6 daysLiquid media
1 day
0.030
0 016
0.023
0.009
Yeast N + methyl ester
0.250
0.042
0.067
0.448
Yeast N + diesel
0.057
2.004
4.700
10.24
Pseu N + methyl ester
0.038
0.073
Pseu N + diesel
0.177
0.147
‘The data reported in the table are the optical densitymeasuredby the spectrophotometerat 520 nm

11

The conclusion of this study of water contamination is that water is a much moresignificant issue for biodiesel than for diesel fuel. Methyl soyate can contain up to 40 times moredissolved water than diesel fuel. In fact, if methyl soyate comes into contact with free water
during transport and storage, which is a virtual certainty, it will absorb 1200- 1500 ppm of thiswater. This is well above the 500 ppm level allowed by the current specification. The greatersolubility of water in methyl soyate may require that the acceptable level of water be raised to1500 ppm The fact that this additional water is bound to the fuel and not available for microbial
growth may justify this increase. The increase in the activity of microorganisms on biodiesel,
especially molds, is also reason for concern, In applications where the fuel is expected to comeinto contact with sufficient quantities of free water to support the growth of microorganisms, thefuel should be treated with a biocide to prevent their growth. This will have an adverse effect on
the biodegradability of the fuel.
3.3 Free Glycerin
Free glycerin, a product from the transesterification reactions, is not present when esters
are washed thoroughly with water. However, glycerin may be present in biodiesel as a result of
inappropriate processing, such as insufficient separation of the glycerin phase or insufficientwashing by water after separation. Glycerin will also be present when distillation is used as theprimary mechanism for its removal. Free glycerin in the fuel is suspected to contribute to deposit
formation in the engine. Since its solubility in methyl esters is low, free glycerin tends to collect,over time, at the bottom of storage tanks. Free glycerin also has a high affinity for water andmonoglycerides. If it is present, it is suspected that it will selectively attract these compounds
from the ester and concentrate them at the bottom of the tank. The objectives of this study were
to determine the solubility of glycerin in methyl esters and the effect of water on the solubility ofglycerin in methyl esters.
Double-distilled methyl esters purchased from Interchem (Kansas City, MO) were mixed
with pure glycerin (Fisher Scientific, Fair Lawn, NJ), and the free glycerin content was measured
by the AOCS Oficial Method Cc 17-79 after 4 hr, 24 hr, and 48 hr of vigorous mixing. To
determine the effect of water on the solubility of glycerin in methyl esters, double-distilled methyl
esters were mixed with a 1: 1 (V/V) mixture of pure glycerin and distilled water. The free glycerol
content in the ester-phase was measured by the AOCS Official Method Cc 17-79 after 4 hr, 8 hr,
48 hr, and 72 hr of vigorous mixing. After 72 hr of mixing, the ester-phase was separated fromthe lower phase and dried on a rotary evaporator to remove the water, and the free glycerin in thedried ester was measured. To determine the effect of the presence of water saturation on thesolubility of glycerin in methyl esters, methyl esters were first saturated with water, then mixed
with pure glycerin for 48 hr, and the free glycerin content was measured.
The results are presented in Tables 8 and 9. The solubility of glycerin in methyl esters wasquite low. Even after 48 hr of mixing, the free glycerin content was less than 0.15%. In thepresence of a large amount of water, the solubility of glycerin in the methyl esters was zero. Thisis because glycerol is more easily dissolved in water than ester. Extending this result to theprocessing of esters, we can assume that during the preparation of methyl esters, the washing step
will wash out all of the free glycerin, if any is present in the ester-phase. When the ester is12Table 8. The solubility of glycerin in methyl esters
Mixing time
4hr

24 hr

48 hr

% free glycerin in ester

0.082

0.144

0.072

Table 9. The effect of water on the solubilityof glycerin in methyl esters

In the presence of a large amount of water

Samples% freeglycerin

4 hr mixing
with water
present

8 hr mixing
with water
present

48 hr
mixing withwater
present

72 hr
mixingwith water
present

72 hr
mixingwith water
removed

0

0

0

0

0

48 hr mixing

previously saturated with water, but no separate water phase is present, the solubility of glycerinin esters may be increased slightly, over that of dry esters from 0.144% to 0.187%.
The free glycerin level allowed by the current specification is 0.02%. This is well below
the solubility of glycerin in the ester but is easily achieved by water washing. Since the primary
problems associated with glycerin are due to its existence as a separate phase, it would appear
that this specification could be relaxed somewhat. A level of 0.05% would probably be
acceptable and might allow alternatives to water washing. Idaho Transtech, Inc. has proposedusing glycerin itself as the washing fluid which would result in higher levels of dissolved glycerinin the final biodiesel product [3].
3.4 Bound Glycerin
Figure 2 shows the relative amounts of monoglycerides (MG), diglycerides (DG), andtriglycerides (TG) that may exist as the amount of alcohol is varied during transesterification [4].
Collectively, these compounds will be referred to as hourzdg/ycerin. Although the data shown
are for sunflower oil and ethanol, the amounts should be similar for soybean oil and methanol.
When 100% excess alcohol is used (2.0 times theoretical) the amount pf unreacted TGs should be
essentially zero. However, some MG and DG may be present. Due to their polarity, thesepartially reacted glycerides should be preferentially attracted to the glycerin phase and removed
when the phases are separated. Their actual concentration in the ester may be less than shown in
the figure.
There are two reasons for keeping the amount of bound glycerin low. First, if the boundglycerin is in solution and is injected into the engine, there is suspicion that it will cause carbon
deposits on the fuel injector tips and piston rings, This suspicion is apparently based on historical0 Ethyl

MOLAR
L
0

I

.33

RATIO

EstersETHANOL / SUNFLOWER

1

I

t

66

I.00

133

ETHANOL( timesFigure 2. Effect of alcohol/triglyceride

theoretical

OIL

I

I

1.66

2.00

i

ratio on transesterification product composition. [4]14experience with trying to use vegetable oils as diesel fuel extenders in the 1970s [5]. The current
specification level of bound glycerin in the NBB specification is 0.24%. This translates, forsoybean oil, into 0.92% MG if all the bound glycerin is present as MG, and I .59% DG if all thebound glycerin is present as DG. The second reason for controlling the amount of bound glycerinis that the partially reacted glycerides, particularly the saturated MGs, have very low solubility inmethyl esters and require high temperatures to keep them from crystallizing.
Table 10 shows the analysis of a creamy paste-like material that was extracted from aplugged fuel filter that had been used with a mixture of 20% biodiesel in 80% No. 1 diesel fuel.
The plugged fuel filter was a final filter located immediately before the fuel fill nozzle at a bus
refueling station in Ames, Iowa. The material was provided by Fosseen Manufacturing andDevelopment, the manager of the biodiesel demonstration project.
The paste consisted of about 25% MGs and DGs. The MGs are over 95% saturated, as
would be expected since the saturated MGs crystallize at higher temperatures than theunsaturated MGs. There were two surprising results associated with this analysis. First, that theDGs were present in almost as large an amount as the MGs. This was unexpected since the DGs
have lower crystallization temperatures than the MGs. Second, that the DG composition is not
skewed toward the saturated species but rather approximates the fatty acid composition of theoriginal soybean oil. At the start of this project it was expected that the principal compounds to
be involved with crystallization would be saturated MGs. However, based on the data presented
in Table 10, the study was expanded to include DGs, and potential interactions between MGs and
DGs
The problem of bound glycerin was addressed by this project in two ways: engine testing
to determine whether the bound glycerin in solution would contribute to engine injector tip
deposits, and laboratory testing to identify the crystallization characteristics of the MGs and DGs.
We did not include TGs in the crystallization study because the MGs and DGs should crystallize
sooner. The engine testing portion of this project has not been completed. This is the portion of
the project that was dependent on a delay in funding The results of this work will be reported at
a later date. This portion of the study focused on the effects of selected pure MGs and DGs on
the crystallization properties of methyl esters, the effect of incomplete transesterification on theamount and composition of MGs and DGs in methyl soyate, and the effect of mixtures of MGs
and DGs on the Tco of methyl soyate.
3.4.1 Crystallizationof Pure Monoglycerides

and Diglycerides

Monoglycerides and diglycerides should be present in methyl soyate in very small amounts
when transesterification is properly executed. However, as noted above, both MGs and DGs were
found in large amounts on plugged fuel filters Due to their high melting points and polar

Table 10. Composition
IsolatesMG
DG

of material removed from plugged fuel filter.
Fatty acid composition% in Fuel Filter Sample
16:0
18:0
18:l
18:2
14%
60.8%
35.9%
1.7%
1.37%
11%
14.9%
6.3%
29.6%
44.5%

18:3
0.15%
4.68%

15
characteristics, MGs and DGs, especially saturated MGs and DGs, may cause crystallization
problems in fuels.
Samples of pure MGs and DGs in methyl ester were prepared by dissolving three MGs(monopalmitin, monostearin, and monoolein) and one DG (dipalmitin) in heated methyl esters.
The esters were Interchem double distilled esters that had been found to have very low levels of
MG and DG.
The effects of the pure MGs and DG on the crystallization of methyl esters are presented
in Table 11, In addition to the crystallization onset temperature, Tco, measured by Differential
Scanning Calorimetry (DSC), the pour point (PP), measured by ASTM standard method D97,
and the cloud point (CP), measured by ASTM standard method D2500, were measured also. The
values in the table are the average of duplicate measurements. The presence of the MGs and DG,
up to 19/o.did not change the pour point of the esters samples. However, the cloud point of thesamples increased with increasing amounts of the saturated MGs or DG, and even the sample with
0.05% saturated MG had a CP significantly higher than that of the control. Dipalmitin had asimilar effect on the crystallization properties of the methyl esters as was found for monopalmitin.
This was surprising because the DGs generally have higher crystallization temperatures.
Monoolein (up to 2%), as expected, had no effect on the CP and PP of the esters. This confirmed
observations made about the fatty acid composition of MGs that were collected from the plugged
fuel filter. About 95% of the MGs in the plugged filter sample were saturated MGs. Unsaturated
MGs seemed not to contribute to the crystallization of the fuels at low temperature.
Determination of Tco from DSC thermograms was difficult (thus, not very reproducible)
for the samples with less than 0.5% MG because the melting-transition peaks of the MGs werevery flat on the baseline in this range. This was especially true for monostearin. For the same
Table 11. Crystallization
mono- or di-glyceride.
I

propertiesof neat methyl esters with various amounts of pure

% MG

1-monopalmitin
1 T ,.. 1 CP 1 PP

esters
2.0
1.0

57.5

-

-

1 47.41 22-3

1 51.6 1 26

0.2

1

-

1

oI

I

-

005
0

I

47

[

1-man ostearinT,
1 CP 1 PP

monoolein
CP 1 PP

dipalmitin
CP 1 PP

10rDGin 1 w> 1 w> 1 (“C) 1 (“C) 1 (“Cl 1 (“Cl 1 (“C) 1 (“C) I (“Cl 1 (“0 1

I
I
I

I

(control)

-

56.5

-

-6

-3

29

-3

1

I

-3

I

3.8

I

7

I

-3

I

I

-3

I

-3

I

-

I

-I

I

-3

I mmT T

_

I

-4
-6

I -3 I

-1

-3

-

-

1 -3
1 -4
-

-3

4.7

-3

-6

-3

-6

I

I

I

-6

-6

1

0

1

-3

I

-3
-3

16

reason, the DSC analysis for T IYowas not very useful for 20% esters blends with less than 1%
MG. However, distinctive MG peaks were not observed with the samples with less than 0.25%
monopalmitin and with less than 0.20% monostearin. Apparently, the cloud of crystals that wasobserved in measuring the CP of the samples has a very low melting enthalpy so that the meltingof the crystals were not detected as a distinctive peak in the DSC thermograms For this reason,
the trends of the CP and TcTo data did not agree well.
3.4.2

Monoglycerideand Diglycerides

FormedDuringIncomplete

Transesterification

Diglycerides are generally observed to have lower crystallization temperatures than MGs.
For example, the p phase of monopalmitin crystallizes at 77°C and the p phase of dipalmitin
crystallizes at 63.5OC [6]. It was surprising to note that monopalmitin and dipalmitin had similareffects on the CP of biodiesel as indicated in Table 11. One explanation for this is that thesaturated DGs are produced in much greater amounts than MGs. To determine the amounts of
MG and DG produced by the transesteritication reactions and whether they were of similar fatty
acid composition to the soybean oil, a series of tests were conducted to determine thecomposition of the MGs and DGs formed during incomplete transesterification.
Different completion levels of transesterification were achieved by conducting theesterification reaction at different soybean oil/methanol ratios (90’/,, 1OO%, 1lOoh, 130%, 150%,
and 200% of the theoretical methanol amount). After the reaction was complete, the methyl esters
were washed with distilled water and vacuum dried. The MGs and DGs in the esters were isolatedon 1-mm silica gel TLC plates, developed with hexene:ethyl ether:acetic acid = 50:50: 1 solvent,and quantitatively analyzed by gas chromatography with heptadecanoic acid methyl ester as aninternal standard.
Table 12 shows the MG and DG contents of esters made at different oil/methanol ratios
and their effects on the cloud point and pour point of the esters. Generally, the MG and DG
decreased with increasing methanol/oil ratio as would be expected from a more complete reaction.
Diglyceride was found in higher concentration than MG in esters made from all incomplete
reactions, but the DGA4G ratio decreased with increasing completion of the reaction, At 200%
theoretical methanol, no MGs and DGs were detected. The pour points of the esters were notaffected at any levels of MG and DG contamination tested. The MG and DG mixture with up to
0 8% MG and 1.69% DG had no effect on the cloud point of the esters. Although MGs and DGs
were detectable in esters made at 130% and 150% theoretical methanol amount, their presence
did not contribute to the crystallization of the esters. The effect of mixtures of MGs and DGs on
the cloud point and pour point of the esters did not seem very great compared with pure MG and
DG since only about 16% of the MGs that existed were saturated, and an even smaller percentageof the DGs were saturated (with two saturated fatty acids). Due to the many possible
configurations of fatty acids in diglycerides, it was hard to extrapolate the results from one type of
pure DG to the mixtures of DGs unless all possible combinations have been studied individually.

Therefore, studying the effect of diglycerides on the crystallization properties of esters is moredifficult than that of monoglycerides.

17
Table 12. Monoglyceride and diglyceride contents of esters made at different oil/methanol
ratio and their effects on the cloud point (CP) and pour point (PP) of esters.

% Theoretical
MeOH amountFatty Acid Profile

150%
DG
MG

10.5

50

23.2

54.0

7.3

(:c9(Y)

3.8

7

-3

3

-3

2.6

2

-3

2.1

-1

-3

2.1

-1

-3

-1

-3

1.69
0 00

200%
DG

DGs I
MGs

0 00

3.4.3 The Effect of DG/MG Ratio on Crystallization
To determine the effect of mixtures of MG and DG that were isolated from soy esters on
the crystallization onset temperature (Tco) of neat esters, 50 g of methyl esters made at 100% of
the theoretical amount of methanol were added to a column packed with 100 g of alumina. One
liter of solvent (ethyl ether:hexane=10:90) was used as the mobile phase to elute the methyl
esters, followed by 1 liter of ethyl ether:hexane=50:50 to elute the DGs (contaminated withmethyl ester). Finally, 500 mL of methanol was used to elute the MGs (contaminated with DGs).
The collected fractions containing MGs and DGs were evaporated, dissolved in a small amount ofchloroform, and applied on l-mm silica gel TLC plates to further purify the MGs and DGs. MGs
and DGs isolated from TLC plates were added to neat methyl esters (from Interchem) at DG/MG
ratios of 2 and 3 and a total DG amount of 1.5%, 2.5%, 3.5%, 4.5%, 5.5%, 6.5%, and 7.5%. A
Perkin-Elmer Series 7 DSC was used to measure the crystallization onset temperature of the18methyl esters. The samples were heated from -7OOC to 90°C at a heating rate of S”C/min. The
Tco was determined as the end point of the highest melting transition, that is, the lowest
temperature at which the sample was completely liquid.
The effect of MG and DG mixtures isolated from soy esters on the crystallization onset
temperature (Tco) of neat esters is presented in Table 13. At a DG/MG ratio of 3.0, a MG
concentration up to 1 5% and DG concentration up to 4.5% had no effect on the Tco of theesters, and the Tco was even slightly lower than that of the control. This may have been causedby the melting point depression effect of the impurities. At higher MGs and DGs concentration, avery small and flat peak indicating a higher melting transition than the methyl esters was observed,
but because the samples were mixtures of MGs and DGs, the peaks were even broader and harder
to distinguish from-the baseline than the pure MG species, as discussed above. At a DG/MG ratioof 2.0, the Tco was significantly increased at all levels of MG and DG compared with the control,
but no differences were found among the different levels. The results suggest the possibility thatthe presence of DGs interferes with the crystallization of MGs. Diglycerides have been reported
to inhibit or retard the phase transition of triglycerides [7,8]. Further studies are needed toconfirm the interfering effect between DGs and MGs.
Table 13. The effect of MG and DG mixtures isolated from soy methyl esters on the
crystallization onset temperature (Tco) of neat esters.
DG/MG ratio,% DG
3.0, 1.5%
3 0, 2.5%
3 0, 3.5O/b
3 0, 4.5%
3.0, 5.5%
3.0, 6.5%
3.0, 7 5%

Actual % MG

Actual % DG

05
1.0
1.1
1.5
1.8
20
2.5

2.0, 1.5%
2 0, 2.5%

0.7
1.3

Tco (“C)

1.5
3.3
35
4.5
5.5
6.5
7.5

Actual DG/MG
ratio
3.0
3.3
3.2
3.0
3.1
3.3
3.0

1.5
2.5

2.1
1.9

28.7
29.2

5.2
4.7
4.4
4.1
16.3
29.2
29.2

19
3.4.4 The effect of bound glycerin on the viscosity of biodiesel and diesel fuelTests were conducted with O%, 0. l%, 0.5%, l%, 2%. 3% and 5% monoglycerides (byweight) added to #l diesel fuel, #2 diesel mel, 20% biodiesel in #l diesel fuel, 20% biodiesel in #2
diesel fuel and 100% biodiesel. A total of 35 samples were prepared. Each sample was heated toabout 70 “C in order to completely dissolve the MGs into the fuel. At room temperature a higher
percent of MG in the fuel makes a very thick gelatinous mixture, but a lower percent of MG
forms crystals in the bottom of the sample. The monoglycerides were derived from soybean oil
but had been hydrogenated. Their composition was: 10 8% monopalmitin, 12.0% monostearin,
76.0% isomers of monoolein, and 1.1% isomers of monolinolein.
After the samples were prepared, each sample was heated to between 40°C and 45°C
before being loaded to the viscometer. The loaded viscometer was then placed in a constanttemperature bath to equalize the sample temperature with the bath temperature. The temperatureof the bath was maintained at 40 (k 0 1) “C The viscosities of the samples are shown in Table 14.
At 40°C, 2%, 3% and 5% MGs and #1 diesel fuel, the mixture forms crystals that separate
from the fuel and plug the viscometer. A similar effect was found for 3% and 5% MG in No. 2
diesel fuel and 5% MG in a 20% biodiesel blend with No. 2 diesel fuel. At 40°C, 0% to 5% MGs
in 100% biodiesel did not form any crystals The viscosity increased as the percentage of MG
was increased for all the fuels tested. However, the increases were not large and, if crystals are
not present, should not affect engine operation.
3.4.5 Discussion of the Bound Glycerin Specification
If all of the 0.24% bound glycerin allowed by the NBB specification were present as MG,
then this would correspond to 0 92% MG. However, in soybean oil-based biodiesel, only about
15% of the MGs are saturated. The data presented in Table 11 indicated that saturated MG
amounts over 0.25% for monopalmitin and 0.2% for monostearin would begin to crystallize
Table 14. Effect of adding monoglycerides on the viscosity of diesel fuels and methyl esters.
Viscosities in cS. All viscosities measured at 40°C.
Fuels blended with monoglycerides

20

1 Crystals

I2881

4 406

1 1.931

1 2.993

I

3.0

Crystals

Crystals

4.405

2.013

3.009

CO

Crystals

Crystals

4.607

2.141

CrystalsI20
according to the crystallization onset temperature. The cloud point indicates that crystals will
form even at saturated MG concentrations as low as 0.05%. If all of the bound glycerin is present
as MG, and the MG composition is the same as for soybean oil, the amount of monopalmitin andmonostearin should be about 0.10% and 0.04%, respectively. This should raise the cloud point of
the fuel by 2-3°C. This would probably not create any problems but any increase in MG beyond
this level is not acceptable.
When mixtures of MGs and DGs were investigated, Table 12 showed that the boundglycerin produced from a transesterification reaction with 130% of the theoretical amount ofmethanol gave 0 73% MG and 1.5 1% DG. This translates into 0.42% total bound glycerin andno change in the cloud point. This suggests that the MG and DG interact in a way that inhibits
crystallization, and that the specification for bound glycerin could be raised However, when thisinteraction was investigated with the data presented in Table 13, it was found that the
crystallization onset temperature depends on the ratio of DG to MG. When 0.7% MG and 1.5%
DG were tested, the Tco was 28.7”C compared with 4.7”C for the baseline ester.
The interaction between MGs and DGs that determines their crystallization properties iscomplex and a complete study was beyond the scope of this project. However, the data presented
here indicate that the level of bound glycerin is close to the limit at which crystallization will occur
and the apparent discrepancies between the CP and T co would need to be resolved before thespecification value should be raised.
3.5 Alcohol
Methyl alcohol is the most common alcohol used for producing biodiesel although ethanol
is used in some cases. Our measurements indicate that when the tranesterification of soybean oil
is conducted with 100% excess methanol, about half of the excess methanol is in solution with theester phase and half is with the glycerin phase. If the biodiesel is washed with water when thetranesterification reaction is complete, there should be very little alcohol left since the alcohol ismore soluble in the water than in the biodiesel. Further, if the biodiesel is vacuum dried toremove the residual water from the washing operation, any remaining methanol should be
removedThe only fuel or engine parameter likely to be affected by the residual alcohol is the flash
point. High concentrations (>5%) of methanol could have an impact on fuel lubricity and cetane
number but low concentrations (<I%) should have no measurable effect, Figure 3 shows the flash
point of biodiesel with varying amounts of methanol. If 100°C is set as the minimum allowable
flashpoint for biodiesel, this will be exceeded by a methanol fraction between 0.18 and 0.22%.
The allowable limit for ethanol is slightly higher at 0.26%. Since there is no known deleterious
effect associated with these small concentrations of methanol, there does not seem to be any
reason to place any restrictions on alcohol except as dictated by the flashpoint restriction. Fuelsuppliers needing to satisfy a 100°C flashpoint specification, the current limit in the National
Biodiesel Board specification will need to get the methanol concentration below about 0.2% andthis should ensure there are no lubricity or cetane number problems associated with highconcentrations of alcohol.

21

180
0

160

E
0
I

140

z

120

zi
z

100

1

5
V

80

E

60

cg

40

2
E

20

~-Methanol(ISU) i--111- Methanol [9];- 0 - Ethanol (ISU) I

0
0

0.2

0.4

0.6

Alcohol

Fraction,

0.8

1

%

Figure 3 The effect of alcohol on the flashpoint of biodiesel
3.6 Free fatty acids, soap, and catalyst
Free fatty acids, soap, and catalyst are treated together because they are related
compounds. Most biodiesel is produced using an alkaline catalyst such as sodium methoxide,sodium hydroxide, or potassium hydroxide. Any free fatty acids that are present will react withthe alkaline catalyst to form soaps. These soaps are then removed during the washing process.
The washing process also removes any residual catalyst.
The primary problem associated with these contaminants is that when the fuel burns in theengine, the metal remaining from the soap or catalyst will be converted to ash. The current
specification for the ash content of biodiesel is 0.02% although this is believed to be an error. It
should be no greater than the 0.01% given by ASTM D 975, the standard for No. 2 diesel fuel.
To investigate the amount of residual catalyst and soap formed during thetransesterification reaction, an experiment was conducted to determine the effectiveness of water
washing A batch of methyl soyate was produced from a food-grade soybean oil using potassiumhydroxide as the catalyst. The methyl soyate was separated from the mixture of methyl soyate,soap and catalyst by washing with distilled water
follows:

The steps of the washing process were as22
1 Add 100 cc of distilled water to a 500 ml separator-y fknel containing approximately 200 cc
of raw methyl soyate (mixture of methyl soyate, soap, catalyst, and unreacted methanol)
Shake the distilled water and the raw methyl soyate mixture.
2 Wait until the soapy water separates from the mixture.
3 Separate the soapy wash water from the mixture and place into a 150 ml flask for later soap
and catalyst tests. The soapy water also contains a portion of the unreacted methanol andcatalyst.
4 Repeat steps l-3 seven times and keep all the wash water samples in 150 ml flasks formeasurement of soap and catalyst.
The amount of soap and residual catalyst in the methyl soyate was determined by adding asample of the wash water to a larger quantity of ethanol and titrating with 0.01N HCl using aphenolphthalein indicator solution. The amount of residual catalyst was calculated from theformula,% Cirtalyst (as potassium hydroxide) = s, l m .l()()
1oooq
where 0, = milliequivalents of 0 OlN HCl added (milliequivalentHCL)(I= sample size in grams= ml of HCL * Normality of

For the soap measurement, 4-5 drops of bromophenol blue indicator solution was added
and the titration continued to the yellow end point. The amount of soap was calculated from theformula:o/0 Soap(as sodirim

ofeate)=Q, 0304.4. loo
lOOOq

where Q2 = milliequivalents of additional 0 OlN HCl added
The percentage of soap and catalyst in the unwashed methyl soyate, wash water, andglycerin are shown in Table 15. The unwashed methyl soyate was found to have 0.149% soap
content and no measurable catalyst. When this methyl soyate was washed, the first batch of washwater (100 cc of distilled water) contains most of the soap and catalyst. The second and the third
wash water contain less soap and no measurable catalyst. The other batches of wash watercontain essentially no soap or catalyst. The level of soap indicated is equal to that measured on
the distilled water used for washing and is probably due to a bias caused by the phenolphthaleinsolution. For the calculations described later, the soap shown for distilled water was subtracted
from the other quantities. Table 15 also shows that most of the soap and catalyst are present in
the glycerin phase.
The methanol present in the glycerin phase can be separated by heating the mixture in a
vacuum. The yield of methyl soyate, glycerin, soap, and a potassium balance are shown in Table

23
Table 15. Percentage of soap and catalyst in wash water, unwashed methyl soyate andTable 16. Total mass of products and potassium balance
Total amount of methyl soyate
170.74 g
Glvcerin and unreacted methanol mixture
30.25 g;Table 17. Composition of glycerin and ester fractions
Glycerin fraction
glycerin
catalystsoapmethanolmethylsoyate
(g)
(g)
(8)
(g)
(8)

Ester fraction
catalyst
soap
w
(g>

19.52

0.015

1.4002

1 183

10.73

170.74methanolw
did not
measure0.254

16. The total amount of the methyl soyateand glycerin produced in the reaction are 170.74 g and
19 52 g respectively.

which is virtually

The total amountthe same as the amountof soap in the seven batchesof soap measuredof washin the unwashedwater

is 0.254 g

methylsoyate.

This

confirms the accuracy of the soap measurements Also, when the potassium portion of the soap
and catalyst are compared to that originally added as catalyst, the agreement is good.
A breakdown of the two phases produced during transesterification is shown in Table 17.
This table shows that the ester fraction contains almost no catalyst and about 17% of the totalsoap produced

during the reaction.

The glycerin

phase contains

almost99% of the residual

24
catalyst The methanol fraction was not measured during this experiment but later testing has
shown that the excess methanol splits fairly evenly between the ester and glycerin phases.
As mentioned earlier, the ash standard for diesel fuel is 0.01%. If this standard is applied
to biodiesel, then it is necessary to get the residual catalyst below 0.015% since potassium makes
up only about 70% of the catalyst mass. The measurements presented here indicate that this levelis achievable by l-2 water washes There does not appear to be any reason for a separate
specification for soap or catalyst, since the ash standard already keeps these contaminants at avery low level. However, the ash standard given in the National Biodiesel Board specification
should be lowered to the ASTM D 975 value of 0.01%.
3.6 Oxidationof Biodiesel

Although not included in the original project description, oxidation was identified during
the course of the project as a major source of biodiesel contamination. This section describes theresults of initial efforts to determine the significance of biodiesel oxidation on overall fuel quality.
Soybean oil has a higher unsaturation level than many other oils and metal catalysts ofoxidation may exist in biodiesel storage tanks, so soydiesel is very susceptible to autooxidation
during storage Upon oxidation, the chemical compositions of methyl esters may be changed, andthis may in turn alter the properties of biodiesel. When this process occurs at ordinary
temperatures, the initial products are hydroperoxides formed by the addition of oxygen to acarbon atom adjacent to a carbon-carbon double bond [IO]. Generally, at this step, the doublebond is left intact, The extent of this level of oxidation can be characterized by the peroxide\*alue As oxidation proceeds, the peroxides may split and form aldehydes and short chain acids.
Oxidation of biodiesel occurs through a series of steps. Most vegetable oils containnatural antioxidants, such as Vitamin E (tocopherol), that inhibit oxidation until the antioxidant isconsumed. When the natural antioxidants are depleted, oxidation proceeds rapidly. The period ofslow oxidation that precedes rapid oxidation is often called the irlductionperiod.
Bailey [lo] states that if two or more double bonds are present in one fatty acid chain,
they have a mzrtrrah’y activating @&f For example, linoleic acid, with two double bonds,
oxidizes more readily than oleic acid with only one double bond and the tendency to oxidize isgreater than can be explained by the increase in the number of double bonds.
In the food industry, the result of oxidation is rancidity which is characterized by an
unpleasant odor. When esters are used as fuel, an important consequence of oxidation is that thehydroperoxides are very unstable and tend to attack elastomers. In addition, the hydroperoxides
can induce polymerization of the esters and form insoluble gums and sediments. Sediment andgum formation can be a problem with diesel fuel also, although the unsaturated nature of soybeanbased biodiesel may aggravate the problem.
Recent engine durability testing with soybean-based biodiesel has shown that biodiesel
may be subject to fuel filter plugging problems caused by sediment and gum formation [ 111. The
currently accepted explanation for the presence of sediment and gum is that the fuel changes
chemically to produce these compounds and this is identified as aJire/ stabi&problem.
Biodiesel’s oxidation is accelerated by heat and light. Generally, when air is present, oxidation
will proceed. The objective of our study was to find the effect of oxidation level on the properties(such as cetane number) of biodiesel

25
When biodiesel has been distilled to remove the high boiling point materials such asmonoglycerides and glycerin, the natural antioxidants such as tocopherol are also removed. In
this project, methyl esters freshly prepared from refined vegetable oil (Flavorite brand) weredistilled under vacuum to remove pigments, polymers, and natural antioxidants. The distillates
were collected from 120°C-1 50°C at less than 0.05 tot-r. Peroxide values (PV) and fatty acidcomposition of the esters were measured just before and after distillation and are shown in Table
18. The fatty acid composition of the esters before and after distillation were similar. Afterdistillation, the PV of the esters was zero. Heating of the esters in the absence of air or oxygenmight have caused the decomposition of the peroxides.
The freshly prepared and distilled methyl esters were oxidized under fluorescent light with
continuous stirring at room temperature. The peroxide value was measured every two days by
the AOCS Official Method. The oxidation curve for distilled and nondistilled methyl esters ispresented in Figure 4. The peroxide value of the freshly prepared, nondistilled esters increased
almost linearly with increasing time during the oxidation period. It took 24 days for these esters
to reach a peroxide value of 80. However, the oxidation of distilled methyl esters was much
faster. It took only 6 days to reach a peroxide value of 96. Distillation apparently removes thenaturally occurring antioxidants without changing the fatty acid composition of the esters.
It was suspected that changing the oxidation level of the biodiesel would have an impact
on the cetane number since peroxide compounds have been proposed as cetane improvers [ 121.
Table 19 shows the results of cetane number measurements on the samples described in Figure 4,
before and after oxidation.
As can be seen in the table, when the freshly prepared esters were oxidized to aperoxide value of 8 1, the cetane number increased from 47.8 to 54.6. A soybean oil with aperoxide value of 81 would generally be considered to be rancid. However, methyl soyate with ahigh peroxide value does not always have the unpleasant smell usually associated with rancidity.
The cetane testing of the distilled esters showed that when the cetane number wasdetermined before oxidation occurred, the cetane number was the same as before distillation. The
decrease from 47 8 to 46.8 is not considered to be significant, However, as with the undistilled
esters, there was a large increase in cetane number as the esters were oxidized, from 46.8 to 55.4.
While the reason for the cetane number increase is not entirely certain, it is likely that thehydroperoxides formed during the oxidation of the esters are responsible. Dialkylperoxides havebeen shown to be strong cetane enhancers [ 121 and hydroperoxides are likely to increase thecetane number also.
To further investigate the effect of oxidation on the cetane number of biodiesel, samples ofmethyl soyate at six different PV levels were prepared. To produce these samples, freshly

Table 18. The peroxide value and fatty acids compositions of soy methyl esters before andafter distillation.
Samples

PV

16.0

18:0

18:1

18:2

18:3

Fresh methyl esters

5.95

10.49%

3 93%

21.66%

55.62%

8.3 1%

Distilled methyl esters

0

9.57%

4.14%

22.03%

55.86%

8.39%

26

100
90

t

80
i

70

f

60

g

50

'g
.

40

I - Undistilled
I--+- Distilled

0

10

~
~

20

Time (days)
Figure 4 Rate of increase of peroxide value for distilled and undistilled esters

prepared, undistilled methyl soyate was mixed in varying amounts with highly oxidized, distilled
methyl soyate Since the previous testing had established that distillation did not affect the cetane
number, any changes in cetane number between these samples were assumed to be due to thedifference in oxidation. Each of the six samples was split into three parts and the 18
testspecimens were submitted as blind samples to a commercial laboratory for cetane testing. The
results of this testing are shown in Figure 5.
Figure 5 shows the three values of cetane number at each of the six levels of peroxide
value. ASTM D613 states that since these measurements were conducted by the same operator,
on the same equipment, and on the same test material, the difference between the highest andlowest value should be within 0.7 to 0.8 cetane numbers. However, the differences in this data
were as large as 3.9. In spite of the experimental variation, the trend toward increasing cetane
number with increasing peroxide value is clear.
The equation for a least-squares-fit cubic polynomial is shown in the figure. The predicted
Table 19. EffectSampleof oxidationand distillation

Freshly prepared methyl esters (PV=6)
Oxidized methyl esters (PV=8 1)
Distilled methyl esters (PV=O)
Distilled and oxidized methyl esters (PV=82)

on cetane number.
Rep 1 Rep 2

Average

46.8
54 6
46.5
55.5

47.8
54.6
46.8
55.4

48.7
54.5
47.2
55.2

27

60

-

$ 55
~50-:pf-d
6 45 --0I40

y = 6E-06x3 -0.0017x2+0.1857xR2 = 0.8446+ 46.17

’

0

20

40

Peroxide

60

80

100

120

Value - meqvlkg

Figure 5. The effect of peroxide value on cetane number.

cetane number for methyl soyate with a peroxide value of zero is 46.17. This value corresponds
well to the low peroxide value cetane number of 47.8 and 46.8 given in Table 19. It appears thatthe cetane boosting effect of the hydroperoxides becomes minimal above a peroxide value of 70.
At the present time, the National Biodiesel Board (NBB) specification does not include
any standard that relates to oxidation. The European standard on which the NBB specification isbased includes a minimum Iodine Value of 115 but this was dropped from the NBB specification
because it prevented the use of soybean oil as a feedstock. The Iodine Value of soybean oil isaround 130 while the Iodine Value of rapeseed oil, the most common feedstock in Europe, is lessthan 115. The Iodine Value is related to the degree of unsaturation of the fatty acids and can be
related to the tendency of the me1 to oxidize. Generally, fuels with more unsaturation and higher
Iodine Values will oxidize more quickly.
A future biodiesel standard should include two additional specifications. It should include
a measure of the biodiesel’s tendency to oxidize. This could be the Iodine Value, or a variation of
an ASTM fuel stability test such as D 2274. The specification should also include a measure of
the current level of oxidation, such as the peroxide value, and a limit should be set on the
maximum PV of the fuel. While additional testing is needed to identify the maximum acceptablevalue of PV for the mel, a PV of 70 may be appropriate since this is the level beyond which thecetane number does not increase. Further increases beyond this level should have only deleterious
effects.

28

4. Conclusions
The following conclusions can be drawn from the work performed in this project:
No specification value is needed for the amount of unsaponifiable matter in biodiesel as longas the level of unsaponifiable matter is no higher than is present in the original feedstock.
The current specification for water content in the NBB specification is not compatible with theexisting diesel fuel distribution and storage network. Biodiesel that is transported or stored inthis system will be in frequent contact with water and will eventually become saturated. Whensaturated, biodiesel can absorb about three times the amount of water allowed by the current
standards for both biodiesel and No. 2 diesel fuel. Either the standard will need to be raised(probably to 1500 ppm) or biodiesel will need to be transported and stored in special, waterfree systems that are separate from those used for diesel fuel.
All biodiesel that will not be used within a short period of its production should be treatedwith a biocide to prevent the growth of microorganisms. These microorganisms, especiallymolds, grow more readily on biodiesel than they do on diesel fuel.
The current specification for free glycerin appears to be adequate and could potentially berelaxed somewhat from 0.02% to 0.05%.
The amount of alcohol in biodiesel is controlled by the specification on the flashpoint. The
current flashpoint limit of 100°C is well above the limits for No. 1 and No. 2 diesel fuels
which are 38°C and 52OC, respectively. The flashpoint limit for biodiesel could be relaxed to52’C, which would allow a higher level of alcohol, without the alcohol rising to level where it
is likely to affect the fuel’s cetane number or lubricity
The crystallization properties of pure monoglycerides suggest that the specification value forbound glycerin is at the limit to prevent crystal formation. However, when monoglycerides
and diglycerides are considered together in ratios consistent with their presence in methyl
esters of soybean oil, the diglycerides appear to inhibit crystal formation by themonoglycerides. Further research into the interactions between the monoglycerides anddiglycerides is needed before an increase in the level of bound glycerin should be considered.
The amounts of free fatty acids, soap, and residual catalyst are not currently included in the
National Biodiesel Board specification. This is acceptable as long as an alkaline catalyst is
used and the biodiesel is water washed. The specification on ash content will keep thesecontaminants at a low level. However, if acid catalysts are used, which is likely if waste
restaurant grease is used as a feedstock, or if an alternative to water washing is used, then aspecification for free fatty acids, soap, and residual catalyst may be necessary.
The specification for biodiesel should include a limit on the tendency of the fuel to oxidize anda limit on the maximum degree of oxidation allowable for use of the fuel in diesel engines.
More research is needed to determine the appropriate tests and limiting values for theseproperties.