UNCONTROLLED IF PRINTED
Carolina BioFuels, LLCDate: 1/22/08
Document Title: Test-Method_CLARUS GC- COLUMN CHANGE/CALIBRATION
Scope and Purpose:
This method describes the column change and calibration procedure for the free and bound glycerin in Biodiesel analysis.
This method can be used for either of the two GC (old Clarus or new Clarus). For explanation purposes, whenever one reads Clarus, one should replace the word “Clarus” with either old Clarus or new Clarus.
Safety:
Gloves must be worn when handling reagents, product or glassware.
Always wear safety glasses.
Review MSDS for the reagents.
GC COLUMN CHANGE PROCEDURE
GC Shutdown:
On TotalChrom menu, press run and clear setup icon.
Click on Run icon followed by “release control”.
Click on Run icon followed by and detach.
Now click on Admin menu, then “cam administration”, click on users, followed by manager.
Highlight item that has “Clarus” in it, and then click on edit menu, followed by remove locks. Make sure that all items with Clarus have been removed.
Now the computer and GC are detached and GC can be operated by itself.
Shut off power for the Clarus GC.
Column Removal:
Hydrogen, helium and air valves back of the GC need to be off.
After GC oven is cooled, remove the old column.
Remove nuts at each end of the column forreuse.
Remove the septum nut and gas line nut.
Remove the injection port glass liner by pushing the thin rod in the injection port cavity from inside the oven.
Glass liner and an “O” ring will come out from the top. Examine the “O” ring, if looks worn out replace it. If glass liner looks dirty, replace it.
Column Installation:
A: Use the same column.
If overall chromatogram is O.K. but some peaks show tailing or not seen, unravel about a turn and a half of column at each end. Cut ~6-12 inches of each end. Install the column into GC as per directions in step C.
B: New Column.
Take out new column from the box. Unravel about a turn of column at each end, cut ~3-4 inches of each end. Install the column into GC as per directions in step C.
C: Install column in GC.
Each end of the column needs to stick out ~10 inches beyond the column cage.
Insert small nut at the thick end of the column, followed by injection port ferrule. Tapered end of the ferrule (PE# 0992-0141) goes first inside the 1/16 inch nut.
Now insert 1/8 inch nut into the thin end of the column. Ferrule (PE# 0990-3981) has a tapered end and small diameter protruded end. Protruded end goes inside the nut first.
Now insert the thicker diameter column with small nut end into injection port.
Insert the thinner diameter column with big nut into detector end.
Loosely tighten both nuts.
Each end of the column should be visible and sticking out on the top of the instrument. If not take column out, unravel some more columns and repeat the steps shown above.
Cut the column on injector side approximately flush with the outside port surface.
Insert glass liner (PE# N610-1539) with “O” ring (PE# 0992-1004) over the column end. Push liner down to make it flush with the outside port surface.
Insert gas line nut and tighten it.
Inside the oven, push the column upward, till you feel resistance. Tighten the nut snugly by hand, followed by approximately ¼ turn by wrench.
Replace septum in the nut and place septum nut on.
On detector end, cut column about ½ inches above the detector surface.
Now push column up about 3 inches, and cut it again.
Pull back the column about 2 inches or so.
Insert wooden dowel in the detector cavity andlet it rest on the detector cavity.
Push column slowly up and see that it pushes the dowel up.
Pull back column so that it does not push dowel, NOW PULL BACK another ~1/2 inches. Tighten the nut snugly by hand, followed by approximately ¼ turn by wrench.
Remove the dowel.
GC Startup:
Turn on all three gases followed by power switch. Logon to GC.
Ignite the flame for the detector.
Go to GC, inject one microliter of heptane and make sure you see big large peak within 30 seconds.
OnTotalChrome software, click on run, followed by attach icon for the Clarus GC.
Click on run, followed by take control icon. Now GC is controlled by computer.
Go to the setup and type in next six lines as standard #5, run # 1, 2,3,4,5 and 6.
Under modify sequence, for the first run (run #1), choose Biodiesel conditioning method. It allows to conditioned column faster and better.
First few runs will have few extra peaks and upward drifts in baseline. As column gets conditioned, baseline will get smoother. Last three runs or so will be consistent.
GC CALIBRATION PROCEDURE
Equipment and Chemicals:
100 uL syringe
ASTM D6584 Standard Solution #5
20 mL vial
Tricaprin, CAS # 621-71-6, (Spectrum Chemical # G3315-02 or equivalent)
Butanetriol,CAS # 42890-76-6, (Spectrum Chemical # B2912-02 or equivalent)
MSTFA reagent, CAS # 24589-78-4 (Spectrum Chemical # M2751-4 or equivalent)
Pyridine, (Spectrum Chemical # HP 812-52 or equivalent)
100 mL volumetric flasks
Analytical Balance
Butanetriol Internal Standard Solution Preparation:
Add accurately approximately 0.0800 gram of Butanetriol in a tarred 100 mL volumetric flask.
Record the weight and calculate the % Butanetriol in the solution. (e.g., 0.0800 gm in 100 mL will give 0.08%).
Add pyridine to the mark.Store the flask in refrigerator.
Tricaprin Internal Standard Solution Preparation:
Add accurately approximately 0.8000 gram of Tricaprin in a tarred 100 mL volumetric flask.
Record the weight and calculate the % Tricaprin in the solution. (e.g., 0.8000 gm will yield 0.8000%)
Add pyridine to the mark. Store the flask in refrigerator.
If Tricaprin is solid (not powder), you can warm the bottle in the fifty degree oven for few minutes only.
GC Standard Preparation:
Take ASTM D6584 Standard Solution #5 ampoule from the refrigerator and break the seal.
Tare the 20 mL vial on the analytical balance and add following solutions
Add 1.0000 +/- 0.005 gram of ASTM D6584 Standard Solution #5
Add 0.1000 +/- 0.005 gram of Butanetriol solution.
Add 0.1000 +/- 0.005 gram of Tricaprin solution
Add 0.1000 +/- 0.005 gram of MSTFA reagent.
Cap the vial, mix it for 30 seconds, and allow vial to stand for minimum of 20 minutes. Label the vial as a Standard #5.
Pull the bottle top dispenser all the way to top, and add full amount (~8 mL) of Heptane into this vial.
Now follow section B though section C of the test procedure of document # 11-02-0007, Test Method_GlycerinAnalysis by GC.
For new GC column, one may have to repeat the analysis up to six times. Last 3-4 runs will be very consistent.
For normal GC validation, single injection of standard may be sufficient.
At the end of analysis time, computer will print out chromatogram and report. Read and record the “% Free Glycerin”, “% Bound Glycerin” and “% Total Glycerin”.
Copy Method File:
When method modification is required, you may copy the method file to the current data file folder or other location (such as desktop) in your computer for future retrieval if needed.
Retention Time Adjustment:
Analyze standard and typical Biodiesel sample and identify each component and their retention time.
Open the Biodiesel method by clicking modify, followed by downloadedmethod icon.
Click on “components” in menu line followed by “edit components”. Click on each component and correct retention time.
Save the method.
Click on run followed by resume sequence icon.
Reprocess the sample file to validate the correct retention time for each component of interest as follows.
Click on batch icon, followed by file and data file.
Click on folder icon and find a file of interest.
For method file choose C:\my methods\clarus\biodiesel.mth
For report file chooseC:\my methods\clarus\expanded report for Biodiesel.rpt
Then click O.K.
Now on menu click on reprocess followed by start.
Report will be sent to printer. Review the report. All the peaks of interest should be correctly identified.
Calibration of Method:
If new column is installed or new internal standard solution is prepared, method may require calibration.
Inject up to 5-6 times standard solution and review each chromatogram.
Last three chromatograms may be more repeatable. Note retention time of each component.
Open the Biodiesel method by clicking modify, followed by downloaded method icon. Update retention time for each component.
Click on components, edit component, and update amount for Butanetriol and Tricaprin. Since we purchase ASTM standard solution 5, other components amount does not require changing.
Save the method.
Click on components icon followed by calibrate.
We will select last three files having good flat baseline and all components identified correctly. We will add these three files in the list. First file will replace the old calibration curve, while other two files will average the calibration.
Now add a standard file to the list, click on “replace”, level “5” and “add”.
Add rest of standard files one at a time by clicking “average” and level “5” and “add’. Finally, click “OK” icon at the bottom.
Save the method. Click on run followed by resume sequence icon.
Now setup a run to test standard #5 again. Review the report. It should read free glycerin, and mono, di and triglycerides amount very close to what you entered in the calibration.
DOCUMENT APPROVAL
Role/Position(Normative) / Owner:
(informational) / Approval Date
QA Manager / Ravi Doshi / 1/28/08
Role/Position / Reviewed By:
CTO / Terry Boone / 1/25/08
EHS / John Roberts / 1/30/08
Approved By:
Document Control / Jan Rector / 1/30/08
REVISION HISTORY
Date / Revision No. / Modification Reason1/22/08 / 00 / Original issue
11-02-0025-00
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