Lipid Analysis by Reverse-Phase HPLC and Corona CAD: Algal Oil

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Corona ® Charged Aerosol DetectorMethod ParametersColumn: *Halo C8, 150 x 4.6 mm, 2.7 µm,at 40°CDetector: Corona CAD PlusNebulizer Heater: OnFilter:NoneMobile Phase A: Methanol/Water/Acetic Acid(750:250:4)Mobile Phase B: Acetontrilie/Methanol/THF/Acetic Acid(500:375:125:4)Gradient Profile: Table 1Flow Rate: 0.8 mL/minRun Time: 72 minutesInjection Volume: 10 µL at 10°CTime %A %B0.0 100 040.0 60 7060.0 10 9065.0 10 9065.1 100 072.0 100 0Table 1. Gradient and Flow Profile.Sample Concentration: 1mg/mL in Methanol/chloroform (1:1)Figure 2. Analysis of Crude Algae Oil Extract* (10μg o.c.) inMethanol/chloroform (1:1) with General Lipid ClassesOutlined.Figure 3. Analysis of Crude Algae Oil Extract* (10μg o.c.)Stripped of Hydrophilic Materials, in Methanol/chloroform(1:1).
The Corona®Charged Aerosol DetectorResults and DiscussionSeveral classes of lipids are identifiable using themethod: free fatty acids, fatty alcohols, phospholipids,mono-, di-, and triacyl-glycerides, sterols and steroids,and fat-soluble vitamins (not shown) are all present ina single chromatogram, as shown in Figure 2. Also,many of these compounds are not visible with UV/Visdetection alone, which makes the Corona CAD theideal detector for these samples. Another benefit liesin the relative uniformity of response of the CAD withdifferent materials: typical response factors lie within20% of each other, making estimates of mass-% frompeak area-% more reliable and accurate than withother means of detection.In developing a process for biofuels from algal oil, oneof the first steps is to remove the hydrophilic material,leaving the more suitable, hydrophobic material forfurther process development. A different sample ofalgal oil that was stripped of hydrophilic material waschromatographed using the same conditions and isshown in Figure 3. This is algal oil is different thanthat depicted in Figure 2, but the load of 10 µg is thesame. As can be seen, the higher amount ofhydrophobic analytes causes the baseline to appearraised. A lower amount of sample injected maylessen this effect.Once the crude material has been transformed into abiofuel, like biodiesel, the Corona CAD can also beused to further characterize the finished product.Application Note #70-8305, “Biodiesel Analysis byNormal Phase HPLC and Corona CAD,” quantifiesremaining free and bound glyceride contaminants thatoccur during base-catalyzed trans-esterification ofvegetable oils.ConclusionsThis application note describes an analytical methodwhich effectively characterizes lipid samplesobtained from algae oil extracts. The Corona CADhas the sensitivity to see low-level compounds forthe researcher or analytical chemist, and it hasreduced chemical requirements (analytes are onlyrequired to not be volatile) to allow for a broadrange of molecular species to be measured. Withthis analysis, it is also possible to generate importantinformation for the process development scientistworking on a robust and well-qualified method. Theuniformity of response for analytes on the CoronaCAD allows for a more accurate picture of analytequantities than other methods of detection.Ordering InformationCorona Plus Charged Aerosol Detector 70-7041Thermal Organizer Module70-5499TANitrogen generator 70-6003Pump, model 584 RS-232** 70-7058Quaternary Low Pressure Gradient 70-5260Autosampler, model 542 70-4152EZChrom Elite (TM) for ESA softwareincluding PC 70-5073*ESA wishes to thank Mac-Mod Analytical, Inc. (Chadds Ford, PA) for theuse of their column and Sapphire Energy (San Diego, CA) for the algal extract.**Modification required - contact ESA Technical Support.ESA Biosciences, Inc.22 Alpha RoadChelmsford, MA 01824-4171USA+1 978.250.7000 Phone+1 978.250.7090 Fax+1 800.959.5095 US Toll freeEmail: info@esainc.comwww.esainc.comESA AnalyticalBrook Farm, Dorton, AylesburyBuckinghamshire HP18 9NHEngland+44 (0)1844 239381 Phone+44 (0)1844 239382 FaxEmail: info@esainc.comwww.esainc.comESA Biosciences, Inc. is an ISO 9001 and 13485 Certified CompanyESA, Corona and CAD are registered trademarks of ESA Biosciences, Inc.The Corona CAD is covered by multiple patents70-8335Rev A
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Lipid Analysis by Reverse-Phase HPLC and Corona CAD: Algal Oil

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