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{{LSApp
  |name        = Biofuels
  |image       = 6584.jpg
  |type        = Sample Prep and Inject
  |id          = Biodiesel ASTM 6584
  |description = Determination of Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatography
}}
{{logo}}
=== Overview ===
Glycerin content can indicate the quality of biodiesel.  Glycerol is formed as a by-product and separated from biodiesel in the production process, however, traces thereof can be found in the final biodiesel product. Glycerin can be in the form of free glycerin or bound glycerin in the form of glycerides. Total glycerin is the sum of these.  A high or excessive content of free and total glycerin can lead to buildup in fuel tanks, clogged fuel systems, injector fouling and deposits on valves and pistons. Unrelated glycerides and free glycerin make the product behave more like soap rather than fuel.

[http://www.astm.org/ American Society for Testing and Materials (ASTM)] created [http://www.astm.org/Standards/D6751.htm '''ASTM Method D6751'''] specifications serve as the benchmark which identifies the standards that pure biodiesel (B100) must meet before being used as a fuel or blended with petrodiesel. There are 14 different required tests according to ASTM D6751-03. The US National Biodiesel Board has adopted the ASTM biodiesel specification and analytical methodology.  ASTM recommends test method ASTM D6584 for the analysis of glycerin in biodiesel fuel by gas chromatography.

[http://www.astm.org/Standards/D6584.htm '''ASTM Method D6584'''] specifies the use of a gas chromatograph (GC) equipped with a cold on-column injector and an FID detector. ASTM D 6584 provides for the quantitative determination of free and total glycerin in B-100 methyl esters by gas chromatography. The range of detection for free glycerin is 0.005 to 0.05 mass%, and total glycerin from 0.05 to 0.5 mass %. This method is not applicable to vegetable oil methyl esters obtained from lauric oils, such as coconut oil and palmkernel oil. ASTM Method D6584 deals with expensive and toxic reagents and requires very complex and time consuming procedures for preparing calibration standards and finished B100 biodiesel. ASTM-6584 requires a two internal standard, five-point calibration curve be generated for free Glycerine, Monoglycerides, Diglycerides and Triglycerides. Manual standards and sample preparation remains a tedious chore that can significantly contribute to errors and lost precision in the GC analysis. Automation can help with all of these. Analyzing the amount of free and total glycerin in the form of mono-, di- or triglycerides during the esterification process is mandatory when manufacturing biodiesel.

[http://en.wikipedia.org/wiki/Gas_chromatography Gas chromatography (GC)] is ideal for measuring important parameters such as total glycerin, fatty acid methyl esters (FAMEs) and methanol levels in biodiesel fuel. Methods like ASTM D-6584 and EN14105 are the standards for testing total glycerin and glycerides in biodiesel oil.

Gas chromatography (GC)can also serve as a tool for process troubleshooting during biodiesel production to ensure trouble-free operation of the fuel in diesel engines. Monitoring the level of free glycerin and any unrelated mono-, di-, and triglycerides will indicate the efficiency and progress of the chemical reaction during the process of making biodiesel.

=== Significant Markets  ===
* Biodiesel Manufactures

* Biodiesel Blenders

* Biodiesel Distributors

* Biofuels CROs

* Fleet Managers
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'''ASTM Method D6584'''

'''9. Calibration and Standardization'''

'''9.2 Standard Solutions''' – Prepare the five standard solutions in ''Table 3'' by transferring the specified volumes by means of microliter [http://www.leapwiki.com/mediawiki/index.php?title=Category:Consumables syringes] to 10mL septa vials
* Add to each of the five standard solutions 100uL of MSTFA
* [http://www.leapwiki.com/mediawiki/index.php?title=Agitator_for_2ml%2C_10ml%2C_20ml_vials Shake] the vial
* Allow the vial to stand for 15 to 20 minutes at room temperature
* [http://www.leapwiki.com/mediawiki/index.php?title=Large_Volume_Dispensing Add approximately 8 mL n-Heptane] to vial
* [http://www.leapwiki.com/mediawiki/index.php?title=Agitator_for_2ml%2C_10ml%2C_20ml_vials Shake] the vial

{| border="1"
|+ '''TABLE 3 Standard Solutions'''
! Standard Solution Number !! 1 !!  2 !!  3 !!  4 !!  5 
|-
! uL of glycerin stock solution
| 10 || 30 || 50 || 70 || 100
|-
! uL of monoolein stock solution
| 20 || 50 || 100 || 150 || 200
|-
! uL of diolein stock solution
| 10 || 20 || 40 || 70 || 100
|-
! uL of triolein stock solution
| 10 || 20 || 40 || 70 || 100
|-
! uL of butanetriol stock solution
| 100 || 100 || 100 || 100 || 100
|-
! uL of tricaprin stock solution
| 100 || 100 || 100 || 100 || 100
|}

'''9.4 Standardization'''<br/>  
'''9.5''' Inject 1 uL of the reaction mixture into the [http://www.leapwiki.com/mediawiki/index.php?title=On-Column_Injection on-column] injection port and start the analysis. 
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'''10. Procedure'''<br/> 
'''10.1''' Weigh to the nearest 0.1 mg approximately 100 mg of sample directly into a 10 mL septa vial. 
* Using microliter [http://www.leapwiki.com/mediawiki/index.php?title=Category:Consumables syringes], add exactly 100 uL of each internal standard and MSTFA. 
* [http://www.leapwiki.com/mediawiki/index.php?title=Agitator_for_2ml%2C_10ml%2C_20ml_vials Shake]the vial
* Allow to set for 15 to 20 minutes at room temperature
* [http://www.leapwiki.com/mediawiki/index.php?title=Large_Volume_Dispensing Add approximately 8 mL n-Heptane] to vial
* [http://www.leapwiki.com/mediawiki/index.php?title=Agitator_for_2ml%2C_10ml%2C_20ml_vials Shake] the vial

'''10.2''' Inject 1 uL of the reaction mixture into the [http://www.leapwiki.com/mediawiki/index.php?title=On-Column_Injection on-column] injection port and start the analysis.
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=== LEAP's Approach ===

[[Image:6584.jpg|600px|Center|System shown with Agilent 7890 and Twin PAL]]

This digital application note describes a LEAP turn-key system for the analysis of free and total glycerines in biodiesel fuel. 
               
LEAP uses a [http://www.leapwiki.com/mediawiki/index.php?title=TwinPAL Twin PAL] to solve this application due to the many different solvent, internal standard, and sample volumes required.

The [http://leapwiki.com/mediawiki/index.php?title=Category:PAL Lower PAL] is the Prep PAL and runs on [http://www.leapwiki.com/mediawiki/index.php?title=Category:Cycle_Composer '''Cycle Composer Software''']. All of the sample prep is performed with this PAL. There is a 100ul [http://www.leapwiki.com/mediawiki/index.php?title=Category:Consumables syringe] for the solvent transfers. There are (7) 2mL [http://www.leapwiki.com/mediawiki/index.php?title=Category:Reagent_Reservoirs solvent locations]. There is a [http://www.leapwiki.com/mediawiki/index.php?title=Large_Volume_Dispensing DISPTool] that is hooked up to a [http://www.leapwiki.com/mediawiki/index.php?title=Dilutor 5 mL dilutor syringe] for the 8 mL Heptane addition. There is a [http://www.leapwiki.com/mediawiki/index.php?title=Category:Fast_Wash_Stationfast wash station] for the through cleaning of the syringe that has two wash solvents. Wash1 is filled with Toluene and Wash 2 is filled with Heptane. There is an [http://www.leapwiki.com/mediawiki/index.php?title=Agitator_for_2ml%2C_10ml%2C_20ml_vials agitator] that is set at 35 deg C and 500RPM to do the mixing. 

The [http://www.leapwiki.com/mediawiki/index.php?title=Category:Combi Upper PAL] is the Inject PAL and runs under ChemStation Software control using the Agilent CTC Control Software. It is using a 5 ul [http://www.leapwiki.com/mediawiki/index.php?title=Category:Consumables syringe] with a tapered needle to do 1 ul [http://www.leapwiki.com/mediawiki/index.php?title=On-Column_Injection on column injection] into the rear injection port. There is a [http://www.leapwiki.com/mediawiki/index.php?title=Category:Fast_Wash_Stationfast wash station] for the through cleaning of the syringe]that has Wash1 is filled with Toluene and Wash 2 is filled with Heptane. 

The expected Sample Prep Time is approximately 26 minutes<br/>
The expected GC Runtime is approximately 31.81 minutes<br/>
The expected GC Cool down time is approximately 7 minutes<br/>

<br/><br/>

=== Software Control ===
The Lower PAL is the ''Prep PAL'' and runs on [http://www.leapwiki.com/mediawiki/index.php?title=Category:Cycle_Composer '''Cycle Composer Software''']. 

The Upper PAL is the ''Inject PAL'' and runs under Agilent ChemStation control.


''This process can be done on other GC's and data systems and does not require the Agilent approach.''

'''Also, LEAP now has the ability use the new [http://www.leapwiki.com/mediawiki/index.php?title=Category:Chronos Chronos Software] as it organizes parallel processes for sample preparation. This utilizes the analysis system to a much greater extent, while observing the time margins of each individual sample preparation. There is a ''single sample list'' for the entire process and integrates well with Xcalibur from Thermo Scientific and ChemStation from Agilent .'''

=== Screenshots ===
[[image:Photo_Icon.png|40px]]
<gallery>
  Image:5stndardsprep.png  | Cycle Composer 5 Standards Prep
  Image:6584SamplePrepISMSTFA.png | Cycle Composer Sample Prep
  Image:CCSTD prep sample list.png  | Cycle Composer Prep Standards
  Image:6584StandardsPrep.png | ChemStation Sequence Table
  Image:SequencetableA.png | ChemStation Sequence Table
  Image:1_ul_inj.png | ChemStation PAL Method for Injection
  Image:GCInjfor6584.png | ChemStation and Cycle for injection
</gallery>

=== Video ===

[[Image:Movie Icon.png|40px]]  [http://www.youtube.com/watch?v=T1K_zCkPkIE Dispense tool in action on youtube]
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===Useful links ===

[[Image:Www icon.png|40px]] [http://www.astm.org ASTM.ORG]

[[Image:Www icon.png|40px]] [http://www.4cleanfuels.com/biofuels/BioDQuality8-07/FQ_workshop-PiedmontBioFuels.pdf An overview of all tests that must be run by Piedmont Biofuels]

[[Image:Www icon.png|40px]] [http://www.chem.agilent.com/cag/prod/GC/Biodiesel_QR.pdf A good general reference site by Agilent]

[[Image:Www icon.png|40px]] [http://biodiesel.engr.uconn.edu/home/Stuart.pdf UCONN.com   A good general reference site on Biodiesel ASTM Methods]

[[Image:Www icon.png|40px]] [http://www.biodiesel.org  A good general reference site on Biodiesel  ]

[[Image:Www icon.png|40px]] [http://www.biodieselmagazine.com/plant-list.jsp Biodiesel Plant Listing ]

[[Image:Www icon.png|40px]] [http://www.biodieselconference.org/2009/default.asp Biodiesel Conference 2009 ]

[[Image:Www icon.png|40px]] [http://www.biodieselconference.org/2010/default.asp Biodiesel Confernece 2010]

[[Image:Www icon.png|40px]] [http://www.nrel.gov/docs/fy07osti/41549.pdf NREL B100 ]

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===More Info===

To get to the main LEAP digital brochure for other applications: [http://www.leapwiki.com/mediawiki/index.php?title=Main_Page LEAP Wiki Main Page]

Info on [[EN 14110 Methanol content by HS]]

Info on [[ASTM D 5453 Sulfur Content]]

[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=TwinPAL More info on the TWIN PAL]
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[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=Category:Accessories More info on LEAP Accessories]
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[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=Large_Volume_Dispensing More info on Large Volume Dispensing]
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[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=Category:Fast_Wash_Station More info on Washing the Syringe]
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[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=Agitator_for_2ml%2C_10ml%2C_20ml_vials More info on Shaking and Agitation]
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[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=Dilutor More info on the Dilutor]
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[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=On-Column_Injection More info on On-Column]
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[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=Category:Reagent_Reservoirs More info on Reagent Storage]
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[[Image:Www icon.png|40px]] [http://www.leapwiki.com/mediawiki/index.php?title=Category:Combi More info on the PAL]
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=== Videos of PAL ===
<br>
[[Image:Movie Icon.png|40px]]
[http://www.youtube.com/user/LEAPTechnologies#g/u LEAP's PAL Application Videos on YouTube]
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[[Image:Chronos_Sample_Sequence.png‎|right]]
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'''Info on Chronos software from LEAP Technologies'''<br>
Chronos Mastersoftware is an ideal solution for automating complex GC Sample Preparation steps on the CTC PAL. Software Integration with data systems ChemStation™ or Xcalibur™ assures seamless functionality with existing systems. Chronos not only exchanges information with other systems, but also controls the complete process and optimizes intelligent time-management resulting in significant improvement in system throughput.Single Sample List Control – including the flexible Twin PAL<br><br><br>
[http://www.leapwiki.com/mediawiki/index.php?title=Category:Chronos Chronos Mastersoftware for organizing parallel processes for sample preparation]
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{{logo}}

LEAP provides automated workstation instrumentation solutions based on the LEAP CTC PAL X, Y, Z syringe only autosampler robot from LEAP Technologies. This extremely flexible, precise, and adaptable liquid handling robotic platform is available in a variety of lengths and options depending on the requirements of your sample preparation and injections for your UHPLC, LC or GC chromatography.LEAP offers full support and service for the PAL platform in addition to being able to write custom macros, cycles, and scheduling to your applications. Please contact LEAP Technologies on how we can help you get maximized throughput with flexible pipetting automation solutions.

=== Contact LEAP ===
{{contact|topic=the PAL and the application}}

=== Keywords ===
biodiesel, free glycerin, gas chromatography with FID, methyl esters, total glycerin, American Society for Testing and Materials (ASTM), product quality, cold on column,  

[[Category: Biofuel]]
[[Category:Application_Solutions]]
[[Category:Cycle Composer]]
[[Category:Gas Chromatography]]
[[Category:Chronos]]