WO2008127617A1

WO2008127617A1 - Method and apparatus for analyzing a sample such as a biodiesel fuel sample

Info

Publication number: WO2008127617A1
Application number: PCT/US2008/004638
Authority: WO
Inventors: Romulus Gaita; Donna Young
Original assignee: Alltech Associates Inc.
Priority date: 2007-04-13
Filing date: 2008-04-10
Publication date: 2008-10-23
Also published as: WO2008127617A9; AR066213A1

Abstract

Methods and apparatus for analyzing a sample, such as a biodiesel fuel sample, are disclosed. The method comprises the steps of determining individual amounts (i) mono-, (ii) di- and (iii) tri-acylglycerols in the biodiesel fuel sample using a liquid chromatography device in combination with a detector and, based on the individual amounts of respective acylglcycerols in the sample, providing value for one or more sample parameters.

Description

METHOD AND APPARATUS FOR ANALYZING A SAMPLE SUCH AS A BIODIESEL FUEL SAMPLE

FIELD OF THE INVENTION

[0001] The present invention is directed to methods and apparatus for analyzing samples such as a biodiesel fuel sample.

BACKGROUND OF THE INVENTION

[0002] Biodiesel, a derivative from plant oils or animal fats, is gaining more attention as an attractive alternative fuel to petroleum diesel due to increased demand for depleting fossil fuel resources, as well as being a more environmentally friendly fuel. [0003] Vegetables oils, such as soybean oil, rapeseed oil, corn oil, palm oil and others, as well as animal fats and recycled greases, are the major sources of biodiesel. Regardless of the feedstock, transesterifϊcation reactions are carried out to produce biodiesel. The transesterification reaction of triacylglycerols (TAGs) in oils is usually done by reacting the TAGs with methanol in the presence of a basic catalyst yielding fatty acid methyl ester (FAME). During the transesterification process, intermediate glycerols such as monoacylglycerols (MAGs) and diacylglycerols (DAGs) are formed. MAGs and DAGs can remain in and contaminate the final biodiesel product. Besides MAGs and DAGs, unreacted TAGs can also be present in and contaminate the final biodiesel product. These contaminants can lead to severe engine problems. Therefore, it is important to have an analytical method to monitor completion of the transesterification reaction, as well as be able to quantify the presence of contaminants in biodiesel at very low levels.

[0004] There is a need in the art for methods of efficiently and effectively analyzing samples such as biodiesel fuel samples. There is also a need in the art for an apparatus capable of effectively analyzing samples such as biodiesel fuel samples, as well as providing analytical data relating to the sample. SUMMARY OF THE INVENTION

[0005] The present invention relates to the discovery of methods for analyzing samples such as biodiesel fuel samples. The disclosed methods provide a number of advantages over known methods of analyzing samples. For example, the disclosed methods of the present invention utilize high pressure liquid chromatography (HPLC) techniques coupled with a detector that identifies and determines amounts of chemical compounds so as to provide quick, reliable detection and quantification of various compounds within a given sample at relatively low content levels (e.g., less than 0.08%). [0006] In the area of biodiesel fuel production, the methods of the present invention can be used to detect, monitor and quantify individual amounts of mono-, di-, and triglycerides within a biodiesel fuel sample. Further, the disclosed methods may be used to monitor and quantify reaction completeness such as completeness of a transesterification reaction in the production of biodiesel fuel. [0007] The present invention is directed to methods of analyzing samples that include glycerols, such as biodiesel fuel samples. In one exemplary embodiment, the method of analyzing a sample comprises the steps of determining individual amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample using a liquid chromatography (LC) device in combination with a detector. Alternatively, the method of analyzing a sample comprises the steps of providing separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample. The method of analyzing a sample may further comprise providing a calculated value for one or more sample parameters selected from the group consisting of a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, and any combination thereof, based on the individual amounts of mono-, di- and tri- acylglycerols in the sample.

[0008] In another exemplary embodiment according to the present invention, the method of analyzing a sample, such as a biodiesel sample, comprises the steps of determining amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample using a liquid chromatography (LC) device in combination with a detector, wherein the liquid chromatography (LC) device provides (1) separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the biodiesel fuel sample, and (2) one or more separate peaks for a biodiesel fuel component of the biodiesel fuel sample. [0009] The present invention is further directed to an- apparatus capable of analyzing a sample such as a biodiesel fuel sample. In one exemplary embodiment, the apparatus suitable for analyzing a sample, such as a biodiesel fuel sample, comprises a sample analyzer that determines individual amounts of (i) mono-, (ii) di- and (iii) tri- acylglycerols in the sample, and computer-executable instructions for calculating a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, or any combination thereof from the individual amounts of mono-, di- and tri- acylglycerols in the sample.

[0010] In another exemplary embodiment according to the present invention, the apparatus capable of analyzing a sample, such as a biodiesel sample, comprises a liquid chromatography device that separates (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; a sample analyzer that provides separate peaks for the total amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; and computer-executable instructions for calculating total amounts of mono-, di- and tri-acylglycerols in the sample.

[0011] In an even further exemplary embodiment according to the present invention includes an apparatus capable of analyzing a sample comprising a liquid chromatography device that separates (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; a sample analyzer that provides separate peaks for the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; and computer-executable instructions for calculating total amounts of mono-, di- and tri-acylglycerols in the sample. The liquid chromatography (LC) device may provide (1) separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample.

[0012] The present invention is even further directed to computer readable medium having stored thereon computer-executable instructions for calculating a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, or any combination thereof, from individual amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in a biodiesel fuel sample. The computer-executable instructions may utilize stored data that correlates individual amounts of mono-, di- and tri-acylglycerols in a biodiesel fuel sample to one or more properties of the biodiesel fuel sample such as a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, or any combination thereof. The computer readable medium may be used to load application code onto an apparatus, such as an apparatus capable of determining individual amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in a biodiesel fuel sample, in order to provide to an operator or user additional information regarding the biodiesel fuel sample being tested.

[0013] These and other features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 depicts an exemplary apparatus of the present invention; and

[0015] FIG. 2 depicts a chromatogram of triglycerides in soy oil using an exemplary apparatus of the present invention; [0016] FIG. 3 depicts a chromatogram of biodiesel fuel contaminated with mono-, di- and tri-acylglycerols using an exemplary apparatus of the present invention;

[0017] FIG. 4 depicts a chromatogram of an 80/20 blend of biodiesel fuel and petrodiesel using an exemplary apparatus of the present invention;

[0018] FIG. 5 depicts a chromatogram of an 80/20 blend of biodiesel fuel and petrodiesel contaminated with mono-, di- and tri- acylglycerols using an exemplary apparatus of the present invention; [0019] FIG. 6A graphically depicts calibration curves of mono- and di-acylglycerols with the y-axis corresponding to Area Counts (mV) and the x-axis corresponding to Concentration (%); [0020] FIG. 6B graphically depicts calibration log curves of mono- and di-acylglycerols with the y-axis corresponding to Log Area Counts (mV) and the x-axis corresponding to Log Concentration (%); [0021] FIG. 7 depicts a chromatogram of biodiesel fuel contaminated with mono-, di- and tri-acylglycerols using an exemplary apparatus of the present invention;

[0022] FIG. 8A graphically depicts a calibration curve of monoacyglycerols with the y-axis corresponding to Area and the x- axis corresponding to Concentration (mg/mL);

[0023] FIG. 8B graphically depicts a calibration curve of diacyglycerols with the y-axis corresponding to Area and the x-axis corresponding to Concentration (mg/mL);

[0024] FIG. 8C graphically depicts a calibration curve of triacyglycerols with the y-axis corresponding to Area and the x-axis corresponding to Concentration (mg/mL);

[0025] FIG. 9A depicts a chromatogram of monoacyglycerol and triacyglycerol standards using an exemplary apparatus of the present invention; and

[0026] FIG. 9B depicts a chromatogram of a diacyglycerol standard using an exemplary apparatus of the present invention.

DETAILED DESCRIPTION OF THE INVENTION [0027] To promote an understanding of the principles of the present invention, descriptions of specific embodiments of the invention follow and specific language is used to describe the specific embodiments. It will nevertheless be understood that no limitation of the scope of the invention is intended by the use of specific language. Alterations, further modifications, and such further applications of the principles of the present invention discussed are contemplated as would normally occur to one ordinarily skilled in the art to which the invention pertains.

[0028] The present invention is directed to methods of analyzing samples including biodiesel fuel samples. The present invention is further directed to an apparatus capable of analyzing samples such as a biodiesel fuel sample. The present invention is even further directed to computer software suitable for use in an apparatus capable of analyzing samples such as a biodiesel fuel sample, wherein the computer software enables the apparatus to perform one or more method steps as described herein. [0029] A description of exemplary methods of analyzing samples and apparatus capable of analyzing samples is provided below.

/. Methods of Analyzing Samples

[0030] The present invention is directed to methods of analyzing a sample such as a biodiesel fuel sample. The methods of analyzing a sample may contain a number of process steps, some of which are described below.

A. Detecting and Quantifying Compounds in a Sample [0031] The following steps may be used to detect the presence and quantify the amount of one or more compounds within a given sample.

1. Compound Separation Step

[0032] The methods of the present invention desirably utilize a liquid chromatography (LC) step to separate compounds within a given sample. Depending on the particular sample, various LC columns, mobile phases, and other process step conditions (e.g., feed rate, gradient, etc.) may be used. Any sample that contains glycerols may be used in the method and apparatus of the present invention, including biofuels such as biodiesel.

[0033] In one exemplary embodiment, the method of analyzing a sample comprises analyzing a biodiesel fuel sample for the presence of monoacylglycerols (MAGs), diacylglycerols (DAGs), and triacylglycerols (TAGs) in the biodiesel fuel sample, either as a group or genus, or as individual species of each group or genus. Any number of commercially available reverse phase LC columns, size exclusion LC columns, gel permeation LC columns, normal phase columns, affinity LC columns, ligand exchange LC columns, and ion- exchange LC column, and the like, may be used on biodiesel fuel samples containing MAGs, DAGs and TAGs. Suitable commercially available columns include, but are not limited to, high purity silica columns commercially available from Grace (Deerfield, IL) under the trade designation Alltech® Alltima® HP Cl 8 HiLoad. [0034] In some exemplary embodiments, LC columns and mobile phases are desirably selected so as to separate MAGs, DAGs and TAGs into single peaks for each class of compounds, as well as separate these single peaks from the peak(s) representing biodiesel fuel components (i.e., fatty acid methyl esters (FAMEs)) of the biodiesel fuel sample on the resulting chromatogram. [0035] A number of LC columns may be used to result in a class- type separation for MAGs, DAGs, TAGs and separate peak(s) for the sample. Suitable columns include, but are not limited to, silica columns, diol columns, polyethylene glycol (PEG) bound columns, and polyvinyl alcohol (PVA) bound columns. Exemplary commercially available silica columns, diol columns, polyethylene glycol (PEG) bound columns, polyvinyl alcohol (PVA) bound columns, cyano bound columns and amino bound columns include, but are not limited to, columns commercially available from Grace (Deerfield, IL) under the trade designation En Vision™ BD, as well as other similar companies.

[0036] A number of mobile phase components may be used to result in separation of MAGs, DAGs, TAGs and the sample. Suitable mobile phase components include, but are not limited to, HPLC grade acetonitrile, ethyl acetate, ethanol, dichloromethane, hexane, methanol, isopropyl alcohol, acetone, chloroform, heptane, ether and combinations thereof. In one desired embodiment, a 50/50 wt% blend of HPLC grade acetonitrile and HPLC grade dichloromethane and subsequent mobile phase gradient is used to allow process monitoring of the sample production, such as in biodiesel production. In another desired embodiment, a 50/50 volume% blend of HPLC grade ethyl acetate and HPLC grade ethanol is used to separate single peaks for each group or genus of the glycerols, such as MAGs, DAGs, TAGs, and separate peak(s) of the remaining components(s) (e.g., FAME in biodiesel) for final quality control testing.

[0037] The feed rate through the LC column is typically less than about 3.0 mL/min, and more typically from about 0.5 mL/min to about 1.5 mL/min, and even more typically, about 1.0 mL/min. [0038] The gradient used in the LC column separation step may vary depending on a number of factors including, but not limited to, the sample being tested, the mobile phase used, the column used, etc. When the sample comprises a biodiesel fuel sample for process monitoring and the mobile phase comprises a 50/50 wt% blend of HPLC grade acetonitrile (A) and HPLC grade dichloromethane (B), one desired gradient is provided in Table 1 below.

Table 1. Exemplary Gradient For Process Monitoring of Biodiesel

Fuel Samples

When the sample comprises a biodiesel fuel sample for final quality control testing and the mobile phase comprises of Ethyl Acetate (A) and Ethanol (B), one desired gradient is provided in Table 2 below.

Table 2. Exemplary Gradient for Final QC of Biodiesel Fuel Samples

2. Detection and Quantifying Step

[0039] The methods of the present invention desirably utilize chemical detectors to detect and quantify compounds separated from one another using the above-described liquid chromatography (LC) step.

[0040] Any number of commercially available detectors, including evaporative light scattering detectors (ELSDs), condensation nucleation light scattering detectors (CNLSDs), charged aerosol detectors (CAD), refractive index detectors (RI), ultraviolet detectors (UV), and mass spectrometric detectors (MS) may be used in the present invention.

B. Providing Calculated Sample Parameter Values [0041] The methods of the present invention may further comprise one or more steps relating to providing one or more calculated values for one or more sample parameters of a given sample to a user or operator. Exemplary sample parameters of a given sample include, but are not limited to, a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, and any combination thereof. A description of possible method steps is provided below.

1. Correlation Step

[0042] The methods of the present invention may comprise a series of steps to produce correlation data between one or more measured sample parameters and one or more calculated (i.e., not measured) sample parameters. For example, in the case of a biodiesel fuel sample, a user may want to know the cetane number of the biodiesel fuel sample in addition to the individual amounts of mono-, di- and tri-acylglycerols in the sample. Instead of having to measure each sample parameter, the methods of the present invention optionally provide one or more calculated sample parameters by simply initiating a trigger mechanism, for example, pushing a button on an apparatus.

[0043] In one exemplary embodiment of the present invention, the method determines individual amounts of mono-, di- and tri- acylglycerols in the sample as described above, and based on the individual amounts of mono-, di- and tri-acylglycerols in the sample, provides a calculated value for one or more sample parameters selected from the group consisting of a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, and any combination thereof. In some exemplary embodiments, the method provides a cloud point, a cetane number, a free fatty acid content value, an iodate number, and a flash point value for a given sample, such as a biodiesel fuel sample. [0044] In order to correlate one or more measured sample parameters (e.g., individual amounts of mono-, di- and tri- acylglycerols in the sample) to one or more calculated values of one or more sample parameters (e.g., a cetane number or a cloud point), standard solutions containing known amounts of one or more compounds (e.g., biodiesel fuel containing known amounts of mono-, di- and tri-acylglycerols) are produced. The standard solutions are then subjected to testing so as to produce a measured sample parameter. For example, biodiesel fuel samples containing known amounts of mono-, di- and tri-acylglycerols can be prepared and tested using conventional test methods to determine a cetane number or a cloud point or a viscosity (or any other sample parameter) for each biodiesel fuel sample.

[0045] It should be noted that any known method of measuring a given sample parameter may be used to generate correlation data. Methods of measuring sample parameters including, but not limited to, a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, and a viscosity, are well known to those skilled in the art, and may be used in the present invention to obtain data correlating to one or more standards containing known amounts of known components. Exemplary methods include, but are not limited to, methods disclosed in ASTM Standard No. D2500 for determining a cloud point; methods disclosed in ASTM Standards Nos. D613-05, D7170-06a, and D6890-07a for determining a cetane number; methods disclosed in ASTM Standard No. D 1500 for determining a color; methods disclosed in ASTM Standard No. D5555-95(2006) for determining a free fatty acid content; methods disclosed in ASTM Standard No. D93-06 for determining a flash point; methods disclosed in ASTM Standard No. D664-06a for determining an acid number; and methods disclosed in ASTM Standard No. D445-06 for determining a viscosity. [0046] The resulting correlation data can be incorporated into correlation tables and electronically stored for use in the methods of the present invention. The stored data may be used by a computing system of an apparatus to correlate, for example, actual measured values of sample parameters, such as actual measured values for individual amounts of mono-, di- and tri-acylglycerols in a given sample, to non-measured values of sample parameters, such as a cloud point, a cetane number, a color, a free fatty acid content, an iodate number, a flash point value, or any combination thereof, for the given sample.

[0047] The stored data may be one component on a computer readable medium and/or may be loaded periodically onto an apparatus that determines individual amounts of mono-, di- and tri-acylglycerols in a given sample. The computer readable medium and apparatus may also have stored thereon computer-executable instructions for performing the method steps of (1) determining individual amounts of mono-, di- and tri-acylglycerols in a given sample and/or (2) providing one or more calculated values of one or more sample parameters for a sample. The stored data may be updated via computer readable medium containing updated stored data and loaded into the apparatus or the stored data can be downloaded directly onto the apparatus from an on-line source (e.g., the Internet).

2. User Interface Steps

[0048] Calculated values of one or more sample parameters may be presented to a user or operator in response to a trigger mechanism. In one exemplary embodiment, a user or operator specifically requests one or more calculated values of one or more sample parameters in a step separate from the above-described step of determining individual amounts of mono-, di- and tri-acylglycerols in a given sample. In this embodiment, a user or operator may push a button on an apparatus, type in a command, or provide any other trigger mechanism so as to initiate the step of providing one or more calculated values of one or more sample parameters.

[0049] In other embodiments, the one or more calculated values of one or more sample parameters are automatically calculated upon initiation of the above-described step of determining individual amounts of mono-, di- and tri-acylglycerols in a given sample. In this embodiment, both the above-described step of determining individual amounts of mono-, di- and tri-acylglycerols in a given sample and the step of providing one or more calculated values of one or more sample parameters occurs in response to a single trigger mechanism, wherein the single trigger mechanism comprises introducing (e.g., injecting) a sample to be tested into an apparatus.

[0050] In some embodiments, it may be desirable to measure or separate and detect a single component and/or chemical specie in a sample, or to measure or separate and detect individual species of a compound class. For example, a particular LC column may separate different species of mono-acylglycerols in a sample. Each specie may be represented on a chromatograph as a single peak. Alternatively, a different LC column may separate compound classes from each other, such as mono-acylglycerols and di-acylglycerols, independent from the number of individual species in the sample. Each compound class is separately detected and shown as single peaks on the chromatograph, or displayed as distinct amounts by the analyzer. [0051] Calculated values of one or more sample parameters may be presented to a user or operator in any format including, but not limited to, a display format (e.g., on a display of an apparatus), a printed format (e.g., a printout generated by an apparatus), an electronic format (e.g., an email or electronic file sent to the user or operator), etc.

C. Other Possible Method Steps

[0052] The above-described methods of the present invention may further comprise any of the following steps.

1. Sampling Step

[0053] The above-described methods of the present invention further comprise a sampling step in which a sample is retrieved from a fluid stream or batch fluid for testing. For example, in one exemplary embodiment, such as in the production of biodiesel fuel, the method may comprise in-line sampling of a fluid stream comprising biodiesel fuel in order to monitor the quality of the biodiesel fuel being produced and/or monitor the transesterifϊcation of TAG in the production of biodiesel fuel, for example, from soy oil. In these monitoring-type embodiments, the method may further comprise providing process control feedback to one or more process variables used to form the fluid stream comprising biodiesel fuel in response to data obtained in an in-line sample analyzing step. [0054] In other exemplary embodiments, the method of the present invention may comprise an "off-line" sampling step, wherein a sample is obtained from a batch process (i.e., a product, such as biodiesel fuel, has already been produced) in order to, for example, verify the quality of the final product, such as a biodiesel fuel product.

2. Comparing Step

[0055] The above-described methods of the present invention may further comprise comparing a detected and quantified amount of one or more contaminants to standards for a given product, and based on the comparison, either accepting or rejecting the given product. For example, in the biodiesel fuel industry, ASTM D6751-07 a sampling step in which a sample is retrieved from a fluid stream or batch fluid for testing. For example, in one exemplary embodiment, such as in the production of biodiesel fuel, the method may comprise in-line sampling of a fluid stream comprising biodiesel fuel in order to monitor the quality of the biodiesel fuel being produced and/or monitor the transesterifϊcation of TAG in the production of biodiesel fuel, for example, from soy oil. In these monitoring-type embodiments, the method may further comprise providing process control feedback to one or more process variables used to form the fluid stream comprising biodiesel fuel in response to data obtained in an in-line sample analyzing step.

3. Updating/Downloading Step

[0056] The above-described methods of the present invention may further comprise a step in which stored data (or other computer- readable instructions for performing one or more method steps) used in the above-described methods is updated to replace an older version of the stored data (or other computer-readable instructions for performing one or more method steps). The step of updating the stored data (or other computer-readable instructions for performing one or more method steps) may comprise inserting a computer- readable medium containing the updated stored data (or other computer-readable instructions for performing one or more method steps) into an apparatus and loading the stored data (or other computer-readable instructions for performing one or more method steps) onto the apparatus from the computer-readable medium. In other embodiments, the step of updating the stored data (or other computer-readable instructions for performing one or more method steps) may comprise downloading the updated stored data (or other computer-readable instructions for performing one or more method steps) onto an apparatus from an on-line source (e.g., the Internet).

//. Apparatus for Analyzing Samples

[0057] The present invention is further directed to an apparatus capable of analyzing a sample such as a biodiesel fuel sample. One exemplary apparatus is shown in FIG. 1. As shown in FIG. 1, exemplary apparatus 10 comprises a mobile phase reservoir 11, an optional degassing system 12, tubing 13 (e.g., NO-OX™ tubing), a HPLC pump 14, an injector system 15, a HPLC column 16, and a detector 17. Any known mobile phase reservoir 11, optional degassing system 12, tubing 13, HPLC pump 14, injector system 15, HPLC column 16, and detector 17 may be used in the present invention such as, for example, those commercially available from Grace (Deerfleld, IL). As discussed above, in desired embodiments of the present invention, detector 17 comprises an evaporative light scattering detector (ELSD), such as the Alltech^® Model 2000ES ELSD or the Alltech^® Model 3300 ELSD both of which are commercially available from Grace (Deerfield, IL).

[0058] One exemplary embodiment according to the present invention includes an apparatus capable of analyzing a sample comprising a liquid chromatography device that separates (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; a sample analyzer that provides separate peaks for the (i) mono-, (ii) di- and (iii) tri- acylglycerols in the sample; and computer-executable instructions for calculating total amounts of mono-, di- and tri-acylglycerols in the sample. The liquid chromatography (LC) device may provide (1) separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample.

[0059] In one exemplary embodiment, the apparatus of the present invention is capable of analyzing a sample, and comprises a sample analyzer that determines amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; and computer-executable instructions for calculating a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, or any combination thereof based on the individual amounts of mono-, di- and tri-acylglycerols in the sample. Typically, the computer-executable instructions utilize stored data that correlates actual measured individual amounts of mono-, di- and tri-acylglycerols in a given sample to a previously calculated value for a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, or any combination thereof of the given sample. This exemplary embodiment is particularly useful when the stored data comprises data relating to biodiesel fuel. [0060] As discussed above, desirably the sample analyzer comprises a liquid chromatography (LC) device in combination with detector. Further, as discussed above, desirably the apparatus comprises a liquid chromatography (LC) device capable of providing (1) single peaks for each group of the (i) mono-, (ii) di- and (iii) tri- acylglycerols in the test sample, and (2) one or more separate peaks for a biodiesel fuel component of the test sample when the sample comprises a contaminated biodiesel fuel sample. [0061] For example, the above-described sample analyzer may be configured to utilize computer-executable instructions for calculating a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, a color, or any combination thereof based on the individual amounts of mono-, di- and tri-acylglycerols in the sample.

[0062] Referring to analyzer 10 in FIG. 1, the apparatus of the present invention may comprise a detector (e.g., detector 17) having numerous buttons 18 and a display 19. Each button 18 may be used to initiate a given function of the apparatus (e.g., exemplary apparatus 10). For example, in one exemplary embodiment, each button 18 may correspond to a given property of a test sample such as a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, or a color of a test sample. By pushing a given button 18, an operator or user can obtain a calculated value for a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, or a color of the test sample. Alternatively, the apparatus may include a personal computer, which utilizes software to perform the above-mentioned functions.

[0063] In other embodiments, detector 17 may comprise button 18 that acts as a single trigger mechanism that, when activated, provides a calculated value for all calculated sample parameters such as a calculated value for each of a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, and a color.

[0064] Detector 17 may further comprise display 19 that visually displays a calculated value for one or more sample parameters selected from the group consisting of a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, a color, and any combination thereof. Display 19 may further visually displays measured values of the amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in the test sample, as well as a total amount of combined (i) mono-, (ii) di- and (iii) tri-acylglycerols in the test sample.

[0065] It should be noted that exemplary apparatus 10 shown in FIG. 1 may further comprise additional components. For example, exemplary apparatus 10 may comprise (i) a display (not shown) separate from (or in addition to) detector 17, and/or (ii) a computing device (e.g., a personal computer) (not shown) separate from (or in addition to) detector 17 so as to provide increased user interface and/or system flexibility to exemplary apparatus 10.

///. Computer Software

[0066] The present invention is further directed to a computer readable medium having stored thereon computer-executable instructions for providing a calculated value for one or more properties of a test sample based on one or more measured values of (i) mono-, (ii) di- and (iii) tri-acylglycerols in the test sample. For example, the computer readable medium may have stored thereon computer- executable instructions for providing a calculated value for a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, a color, or any combination thereof of a given test sample based on one or more measured values of individual amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in a biodiesel fuel sample.

[0067] The computer-executable instructions typically utilize stored data that correlates individual amounts of mono-, di- and tri- acylglycerols in a given sample to a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, a color, or any combination thereof of the given sample. As discussed above, the computer readable medium may be used to load stored data and/or application code onto an apparatus so as to perform one or more of the above-described methods.

IV. Applications /Uses [0068] The above-described methods, apparatus and computer software may be used to (1) detect the presence of one or more compounds in a variety of samples, and optionally (2) provide one or more calculated values for one or more properties (e.g., flash point) of a given sample. The above-described methods, apparatus and computer software find applicability in any industry that utilizes HPLC including, but not limited to, the petroleum industry, the pharmaceutical industry, analytical labs, etc.

[0069] In one desired embodiment, the above-described methods and apparatus of the present invention are used in the biodiesel industry. The above-described methods and apparatus may be used to (1) monitor the transesterification of TAG in the production of biodiesel fuel, (2) monitor the quality of biodiesel fuel being produced, and/or (3) testing the quality of the biodiesel fuel after production.

[0070] In one exemplary embodiment, the above-described methods and/or apparatus are used in a method of monitoring the transesterification of TAG in the production of biodiesel fuel. Such a method may comprise one or more of the following steps: (1) bringing degummed triglycerides into contact with a plurality of silica particles to reduce an amount of phosphorus within the degummed triglycerides so as to form phospholipids-deficient triglycerides typically having from greater than about 1 ppm to about 10 ppm phosphorus; (2) separating the phospholipids-deficient triglycerides from the plurality of silica particles to form a silica-free triglyceride product; and contacting the silica-free triglyceride product with a stripping medium to reduce an amount of free fatty acids within the silica-free triglyceride product so as to form a biodiesel fuel precursor typically having less than about 0.20% wt% free fatty acids based on a total weight of the biodiesel fuel precursor; and converting the biodiesel fuel precursor into a biodiesel fuel using the transesterification step. Other possible method steps in the production of biodiesel fuel include those disclosed in U.S. Provisional Patent Application No. 60/777,303, entitled "PHYSICAL REFINING PROCESS USING ADSORBENT PARTICLES FOR THE PRODUCTION OF BIODIESEL FUEL" AND FILED ON FEBRUARY 28, 2006, the subject of which is hereby incorporated by reference in its entirety. [0071] The above-described methods and/or apparatus are particularly useful in methods of monitoring the transesterification of TAGs in the production of biodiesel fuel, and other methods, due to the relatively fast run speeds and the ability to detect and quantify low levels of contaminates. For example, run times of less than 35 minutes are typical using the above-described LC technique in combination with a detector. Further, detection of contaminant levels as low as 0.08% is typical using the above-described LC technique in combination with such a detector.

EXAMPLES

[0072] The present invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

Example 1 :

[0073] The reliability and sensitivity of an apparatus according to the present invention comprising a reverse phase HPLC system and an ELSD is tested using the following instrumentation, column, reagents, samples and procedures.

Instrumentation:

[0074] Agilent® (Wilmington, DE, USA) 1 100 HPLC system

Alltech® (Deerfield, IL, USA) Model 2000ES ELSD

Column:

[0075] Alltech® Alltima™ HP Cl 8 Hi-Load, 5 μm, 250 x 4.6 mm (Part No. 87698)

Mobile Phase: A: Acetonitrile

B: Dichloromethane Flow rate: 1.0 ml/min Detector settings: Imp OFF, 75C, 2.0 L/min Reagents and Samples:

HPLC grade acetonitrile and HPLC grade dichloromethane

(Burdick and Jackson (Muskegon, MI))

Mono-, Di, and Triacylglycerides standards (Sigma- Aldrich (St.

Louis, MO))

Fatty Acid Methyl Esters (Sigma- Aldrich (St. Louis, MO))

Soy Oil (Whole Foods (Deerfield, IL))

Standard and Sample Preparation:

Soy Oil

[0076] 100 μL of soy oil was diluted in a volumetric flask to 100 ml with 50/50, Acetonitrile/Dichloromethane

Standards Preparation

[0077] 1.0 mg/ml standards of mono-, di and triacylglycerides were prepared in 50/50, Acetonitrile/Dichloromethane

Biodiesel Preparation (BlOO)

[0078] A biodiesel sample was artificially prepared to simulate biodiesel from soy oil by mixing the following volumes: 54 ml Methyl Linoleate (54%), 20 ml Methyl Oleate (20%), 16 ml Methyl Palmitate (16%), and 10 ml Methyl Stearate (10%).

Contaminated Biodiesel Preparation (BlOO)

[0079] In a 50 ml volumetric flask, the following amounts were added and diluted to 50 ml with artificial biodiesel: 0.050 g 1- Linoleyl-rac glycerol (1-L), 0.050 g 1 -Oleoyl-rac-glycerol (l-O), 0.050 g 1-Stearoyl-rac-glycerol (1-S), 0.050 g 1,3-Dilinoleyl-rac- glycerol (1 ,3-LL) and 0.200 g soy oil yielding total impurities of 0.8%.

Contaminated 80/20 Petrodiesel/Biodiesel Preparation, B20 [0080] A blend of petrodiesel and Contaminated BlOO biodiesel was prepared by mixing 80% petrodiesel and 20% Contaminated BlOO biodiesel. Sov Oil Test

[0081] A 5μL injection of 1.6 mg/ml soy oil in 50/50

Acetonitrile/Dichloromethane was injected into the above-describe apparatus shown above using the following gradient:

[0082] As shown in FIG. 2, the resulting chromatogram clearly depicted the triglycerides in the soy oil using the methods and apparatus of the present invention. The triacyglycerol content and composition in the soy oil (or other oils) was easily determined using the above-described method and apparatus.

Contaminated Biodiesel Test

[0083] A 5μL injection of contaminated biodiesel (BlOO) diluted in 50/50 Acetonitrile/Dichloromethane (contaminated with 0.01% 1-L, 0.01% l-O, 0.01% 1-S, 0.01% 1,3-LL and 0.04% of soy oil resulting in a total contamination of 0.08%) was injected into the above- describe apparatus shown above using the following gradient:

[0084] As shown in FIG. 3, the resulting chromatogram clearly depicted the peaks for the contaminants in the biodiesel sample using the methods and apparatus of the present invention. Further, the resulting chromatogram clearly depicted peaks for the unreacted soy oil in the biodiesel sample. The contaminant content and composition in the biodiesel sample was easily determined using the above- described method and apparatus.

Contaminated 80/20 Petrodiesel/Biodiesel Test [0085] A 5μL injection of an 80/20 blend of biodiesel fuel (BlOO) and petrodiesel diluted in 50/50 Acetonitrile/Dichloromethane was injected into the above-describe apparatus shown above using the following gradient:

[0086] As shown in FIG. 4, the resulting chromatogram clearly- depicted peaks for the biodiesel component of the sample using the methods and apparatus of the present invention. Further, the resulting chromatogram showed that the petrodiesel component was volatized and not detected by the ELSD.

Contaminated 80/20 Petrodiesel/Biodiesel Test

[0087] A 5μL injection of an 80/20 blend of contaminated biodiesel fuel (BlOO) and petrodiesel (contaminated with 0.01% 1-L, 0.01% l-O, 0.01% 1-S, 0.01% 1,3-LL and 0.04% of soy oil resulting in a total contamination of 0.08%) was injected into the above- describe apparatus shown above using the following gradient:

[0088] As shown in FIG. 5, the resulting chromatogram clearly depicted (i) the peaks for the contaminants in the biodiesel sample, (ii) peaks for the unreacted soyoil in the biodiesel sample, and (iii) negligible interference from the petrodiesel component of the sample. The contaminant content and composition in the sample was easily determined using the above-described method and apparatus. The petrodiesel component was volatized and not detected by ELSD, which eliminated potential interferences. The less volatile FAMEs, mono-, di- and triacylglycerol were detected. The FAMEs also indicated which feedstock was used to produce the B20 composition. [0089] The contaminated synthetic biodiesel BlOO was used to establish calibration curves and limits of quantitation by performing serial dilutions. A good correlation was obtained for all the impurities as shown in FIG. 6A. [0090] By nature, evaporative light scattering detectors (ELSDs) are non-linear; however, the calibrations curves shown in FIG. 6A were made to have a linear fit by plotting log area counts vs. log concentrations as shown in FIG. 6B. The limit of quantitation was also determined for each mono- and diacylglycerol as shown in Table 3 below. The total impurities quantitation of mono-, di and triacylglycerols was determined to be 0.09%, which is lower than the allowable 0.1%.

Table 3. Limits of Quantitation For Mono- and Diacyl glycerols

Example 2:

[0091] The accuracy, precision and sensitivity of an apparatus comprising a size exclusion HPLC method and HPLC system with an ELSD is tested using the following instrumentation, columns, reagents, samples and procedures.

Instrumentation:

[0092] Grace (Columbia, MD, USA) Ensight Biodiesel Analyzer

Alltech (Deerfield, IL, USA) 3300 ELSD

Agilent (Wilmington, DE, USA) EZ Start software Columns:

[0093] EnVision™ BD, 7um, 250 x 4.6mm (x3)

Mobile Phase: Ethyl Acetate : Ethanol (50:50)

Flowrate: 0.8mL/min

Detector Settings: 4OC, 1.5L/min

Reagents and Samples:

[0094] HPLC grade ethyl acetate (Sigma (St. Louis, MO))

HPLC grade ethanol (Burdick and Jackson (Muskegon, MI))

Mono-, di- and triacylglyceride standards (Nu-Chek Prep

(Elysian, MN))

Fatty acid methyl esters (Sigma (St. Louis, MO))

Soy oil (Whole Foods (Deerfield, IL))

Standard Preparation:

[0095] Individual stock solutions of MAGs and DAGs in ethyl acetate are prepared.

MAGs

[0096] Each MAG is weighed out and diluted with 1OmL of ethyl acetate.

Methyl palmitate - 0.0108g =

10.8mg/10.00mL=1.08mg/mL

Methyl stearate - 0.0106g =

10.6mg/10.00mL=l .06mg/mL

Methyl oleate - .031Og = 31.0mg/10.00mL= 3.10mg/mL

Methyl linoleate - 0.0744g

=74.4mg/10.00mL=7.44mg/mL

Methyl linolenate - 0.0115g = 11.5mg/mL/10.00mL

=1.15mg/mL

[0097] Further dilutions are made for methyl oleate and methyl linoleate so their concentrations were about l. lmg/mL.

Methyl oleate - 3.10mg/mL1.80mL/5.00mL=1.12mg/mL

Methyl linoleate - 7.44mg/mL

1.50mL/l 0.0OmL=I .12mg/mL [0098] A subsequent stock MAG blended solution is prepared mimicking the proportions of total fatty acids in soybean oil to achieve the best quantitative accuracy.

[0099] Volume of 1. lmg/mL MAG standard is used to make blended solution:

Methyl palmitate - 1.1 OmL

Methyl stearate - 0.4OmL

Methyl oleate - 2.4OmL

Methyl linoleate - 5.4OmL

Methyl linolenate - 0.7OmL

The concentration of the blended solution used to make the calibration level standards is 1.1 lmg/mL.

DAGs

[0100] For the individual DAG stock solutions, a 50:50 ratio is used for each isomer of the appropriate diacylglycerol.

[0101] Each DAG is weighed out and diluted with 1 OmL of ethyl acetate.

Diethyl palmitate - 0.0206g =

20.6mg/l 0.00mL=2.06mg/mL

Diethyl stearate - 0.0209g =

20.9mg/l 0.00mL=2.09mg/mL

Diethyl oleate - .024Og = 24.0mg/10.00mL= 2.40mg/mL

Diethyl linoleate - 0.0246g

=24.6mg/10.00mL=2.46mg/mL

Diethyl linolenate - 0.0245g =

24.5mg/mL/10.00mL=2.45mg/mL

[0102] A subsequent stock DAG blended solution is prepared mimicking the proportions of total fatty acids in soybean oil to achieve the best quantitative accuracy. [0103] Volume of DAG standard used to make blended solution:

Methyl palmitate - 1.1 OmL

Methyl stearate - 0.4OmL

Methyl oleate - 2.1OmL

Methyl linoleate - 4.6OmL

Methyl linolenate - 1.2OmL

Ethyl Acetate - 1.2OmL

[0104] The concentration of the blended solution used to make the calibration level standards is 2.09mg/mL.

TAGs [0105] A blended TAG solution is made by simply diluting soybean oil.

0.1065mg/mL = 106.5mg/10mL=10.65mg/mL

[0106] Using the MAG, DAG and TAG blended solutions, five calibration level standards are prepared. Following are the concentration levels of the standards in mg/mL.

Sample Preparation:

Two biodiesel samples used to test method reproducibility are provided by Missouri Better Bean (Bunceton, MO) and are diluted with ethyl acetate. The resulting concentrations are 10.27mg/mL of biodiesel sample 1 and 10.17mg/mL of biodiesel in sample 2.

Synthetic biodiesel

[0107] A mix of pure FAMEs is prepared mimicking the proportions of total fatty acids in soybean oil. FAMEs are weighed out as follows:

Methyl Ester of Palmitic Acid - 1.1058g

Methyl Ester of Stearic Acid - 0.4018g

Methyl Ester of Oleic Acid - 2.403Og

Methyl Ester of Linoleic Acid - 5.3985g

Methyl Ester of Linolenic Acid - 0.6986g

0.0577g of the FAME mix is added to a 5mL solution containing 0.1557mg/mL MAGs, .251 lmg/mL DAGs, and 0.4686mg/mL TAGs.

[0108] 2OuL of synthetic biodiesel is injected into the above- described apparatus. As shown in FIG. 7 the resulting chromatogram clearly depicted the class-type separation with separate single peaks for MAGs, DAGs and TAGs in biodiesel. Using the methods and apparatus of the present invention simplifies total glycerin quantitation compared to ASTM method D6584. Since the glycerides are separated by class into single peaks only three peaks need to be quantified versus 15 or more individual glyceride peaks depending on the feed stock.

Quantitative Analysis:

[0109] The MAGs, DAGs and TAGs standards are used to establish calibration curves. A good correlation is obtained for all the glycerides as shown in FIG. 9 A, 9B and 9C. The external standard calibration curves contribute largely to the accuracy of the quantitative results. A calibration curve is most reliable when its R-squared value is at or near 1. The correlation coefficients, R-squared, for the glycerides are 0.998 or better.

[01 10] Quantitative determination of MAGs, DAGs and TAGs in the synthetic biodiesel is performed to verify the accuracy of the

HPLC method. Two replicate injections of the synthetic biodiesel are run.

[0111] The quantitative results are calculated using the calibration curves. The relative error is 4% or better for these results, proving the method is highly accurate. Table 4 shows the results and relative error.

Table 4.

Sensitivity Determination:

[0112] The sensitivity of the method and system surpasses that required of ASTM method D6584 for glycerides. FIG. 1OA and 1OB show low level concentrations, 0.00625% MAG, 0.0238% TAG and 0.00580% DAG standards, respectively. Precision Testing:

[0113] In order to check the precision of the instrument and the method, three samples of biodiesel are injected 15 times into the HPLC. The %RSD data is shown in the Table 5. The data shows excellent reproducibility of the instrument and method.

Table 5. Reproducibility of Glyceride Standards

%RSD

[0114] While the invention has been described with a limited number of embodiments, these specific embodiments are not intended to limit the scope of the invention as otherwise described and claimed herein. It may be evident to those of ordinary skill in the art upon review of the exemplary embodiments herein that further modifications, equivalents, and variations are possible. All parts and percentages in the examples, as well as in the remainder of the specification, are by weight unless otherwise specified. Further, any range of numbers recited in the specification or claims, such as that representing a particular set of properties, units of measure, conditions, physical states or percentages, is intended to literally incorporate expressly herein by reference or otherwise, any number falling within such range, including any subset of numbers within any range so recited. For example, whenever a numerical range with a lower limit, R_L, and an upper limit Ru, is disclosed, any number R falling within the range is specifically disclosed. In particular, the following numbers R within the range are specifically disclosed: R = R_L + k(R_y -R_L), where k is a variable ranging from 1% to 100% with a 1% increment, e.g., k is 1%, 2%, 3%, 4%, 5%. ... 50%, 51%, 52%. ... 95%, 96%, 97%, 98%, 99%, or 100%. Moreover, any numerical range represented by any two values of R, as calculated above is also specifically disclosed. Any modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All publications cited herein are incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of analyzing a sample, said method comprising the steps of: determining individual amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample using a liquid chromatography (LC) device in combination with a detector.

2. The method of Claim 1, further comprising: based on the individual amounts of mono-, di- and tri- acylglycerols in the sample, providing a calculated value for one or more sample parameters selected from the group consisting of a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, and any combination thereof.

3. The method of Claim 2, wherein said providing step provides a cloud point, a cetane number, a free fatty acid content value, an iodate number, and a flash point value for the sample.

4. The method of Claim 1, wherein the liquid chromatography (LC) device provides separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the biodiesel fuel sample.

5. The method of Claim 1, wherein said determining step occurs in response to a trigger mechanism.

6. The method of Claim 1, wherein said detector comprises light scattering detectors (ELSDs), condensation nucleation light scattering detectors (CNLSDs), charged aerosol detectors (CAD), refractive index detectors (RI), ultraviolet detectors (UV), and mass spectrometric detectors (MS).

7. The method of Claim 1, wherein the sample comprises a biodiesel fuel sample.

8. The method of Claim 7, wherein the liquid chromatography (LC) device provides (1) separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the biodiesel fuel sample, and (2) one or more separate peaks for a biodiesel fuel component of the biodiesel fuel sample.

9. The method of Claim I₅ further comprising: in-line sampling of a fluid stream in order to obtain the sample.

10. The method of Claim 9, further comprising: in response to data obtained in said determining step, providing process control feedback to one or more process variables used to form the fluid stream.

1 1. A method of analyzing a sample, said method comprising the steps of: determining amounts of (i) mono-, (ii) di- and (iii) tri- acylglycerols in the sample using a liquid chromatography (LC) device in combination with a detector; wherein the liquid chromatography (LC) device provides (1) separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the biodiesel fuel sample.

12. An apparatus capable of analyzing a sample, said apparatus comprising: a sample analyzer that determines amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; and computer-executable instructions for calculating a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, or any combination thereof from the individual amounts of mono-, di- and tri- acylglycerols in the sample.

13. The apparatus of Claim 12, wherein the computer-executable instructions utilize stored data that correlates individual amounts of mono-, di- and tri-acylglycerols in a given sample to a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, or any combination thereof of the given sample.

14. The apparatus of Claim 13, wherein the stored data comprises data relating to biodiesel fuel.

15. The apparatus of Claim 12, wherein the sample analyzer comprises evaporative light scattering detectors (ELSDs), condensation nucleation light scattering detectors (CNLSDs), charged aerosol detectors (CAD), refractive index detectors (RI), ultraviolet detectors (UV), or mass spectrometric detectors (MS).

16. The apparatus of Claim 12, wherein the sample analyzer comprises a liquid chromatography (LC) device in combination with an evaporative light scattering detector (ELSD).

17. The apparatus of Claim 12, further comprising: a single trigger mechanism that, when activated, provides a calculated value for one or more sample parameters selected from the group consisting of a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, and any combination thereof.

18. The apparatus of Claim 12, further comprising: a single trigger mechanism that, when activated, provides a cloud point, a cetane number, a free fatty acid content value, an iodate number, and a flash point value of the sample.

19. The apparatus of Claim 12, further comprising: a display that visually displays a calculated value for one or more sample parameters selected from the group consisting of a cloud point, a cetane number, a free fatty acid content value, an iodate number, a flash point value, an acid number, a viscosity, and any combination thereof.

20. The apparatus of Claim 13, wherein the liquid chromatography (LC) device provides (1) separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample, and (2) one or more separate peaks for a biodiesel fuel component of the sample when the sample comprises a contaminated biodiesel fuel sample.

21. An apparatus capable of analyzing a sample, said apparatus comprising: a liquid chromatography device that separates (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; a sample analyzer that provides separate peaks for the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample; and computer-executable instructions for calculating total amounts of mono-, di- and tri-acylglycerols in the sample.

22. The apparatus of Claim 21, wherein the liquid chromatography (LC) device provides (1) separate single peaks for each group of the (i) mono-, (ii) di- and (iii) tri-acylglycerols in the sample.

23. A computer readable medium having stored thereon computer- executable instructions for calculating a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, or any combination thereof from individual amounts of (i) mono-, (ii) di- and (iii) tri-acylglycerols in a biodiesel fuel sample.

24. The computer readable medium of Claim 23, wherein the computer-executable instructions utilize stored data that correlates individual amounts of mono-, di- and tri-acylglycerols in a given biodiesel fuel sample to a cloud point, a cetane number, a free fatty acid content, an iodate number, a flash point value, an acid number, a viscosity, or any combination thereof of the given biodiesel fuel sample.