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lecithin Determinationof AOCSOfficialMethodJa7b

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AOCS Official Method Ja 7b-91（Reapproved 1997）Determination of Lecithin Phospholipids by HPLC.rar,lecithin,Determinationof,AOCSOfficialMethodJa7b

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Page 1 of 2 SAMPLING AND ANALYSIS OF LECITHIN AOCS Official Method Ja 7b-91 Reapproved 1997 Determination of Lecithin Phospholipids by HPLC DEFINITION This method is for the direct determination of single phospholipids (PE, phosphatidylethanolamine; PA, phosphatidic acid; PI, phosphatidylinositol; PC, phosphatidylcholine) in lecithin by high-perfor- mance liquid chromatography (References, 1). SCOPE Applicable to oil-containing lecithins, deoiled lecithins, lecithin fractions; not applicable to lyso-PC and lyso-PE. APPARATUS 1. HPLC basic equipment (a) Sample feeding systemwith sample loop, corre- sponding to the injection volume (10 L). (b) High-pressure pump. (c) UV detectorfor determination at 206 nm. (d) Suitable integrator. (e) HPLC sep a ration columnMicro p a rt i c u l ate spheri c a l silica, 5-m part i cle size, 200 4.6 mm i.d. (Alltech A s s o c i ates, Inc., Deerfi e l d, IL, USA), or equiva l e n t . 2. Microliter syringe25 L, graduated in 1 L. 3. Volumetric flasks10, 500, and 1000 mL. REAGENTS 1. n-HexaneHPLC grade (see Notes, Caution). 2. 2-PropanolHPLC grade. 3. WaterHPLC grade. 4. Sodium acetate 3H2Oreagent grade. 5. Acetic acidreagent grade. 6. Phospholipid standardsPE (#P5274), PA (#P2767), PI (#P0639), PC (#P6263); ava i l able from Sigma Chemical Co., St. Louis, MO, USA. 7. 0.2 M dodium acetate solutionprep a red by dissolv- ing 27.22 g sodium acetate 3H2O in 1000 mL HPLC- grade water. 8. 0.2 N acetic acidprep a red by dissolving 6.005 g glacial acetic acid in 500 mL HPLC-grade water. 9. Walpole acetate buffer (pH 4.2)prepared by mixing 73.5 mL 0.2 N acetic acid (Reagents, 8) to 26.5 mL 0.2 M sodium acetate solution (Reagents, 7), adjusted to exactly pH 4.2 with the aid of a pH meter. 10. Mobile phasen-hexane/2-propanol/acetate buffer pH 4.2 (8:8:1, v/v/v). PROCEDURE 1. P re t re atment of columnTo ach i eve a stable sep a rat- ing performance of the column (see Notes, 1), it must be charged with a certain quantity of the buffer. This is done by rinsing the column for at least 3 days with the mobile phase at a fl ow rate of 0.2 mL/min. Then a s ep a ration of the baseline and a stable retention time should be achieved. 2. After conditioning the column, flow rate is adjusted to 2 mL/min. 3. C a l i b ration curve To prep a re the calibration curve, dissolve 10, 20, 30 and 40 mg of each single phospho- lipid in 10 mL of the mobile phase. Prepare four solu- tions of mixed phospholipid standards. N o t e The solution time may re q u i re up to 3 hrs . Accurately inject 10 L of these calibration solutions. Plot the peak areas against mass in a graph. 4. Sample pre t re at m e n t D i s s o l ve 50250 mg of the lecithin in 10 mL of the mobile phase (see Notes, 2). Prep a re in the same way as the calibration substances. Inject ex a c t ly 10 L of the sample solution into the column. 5. Rinsing of pump head/systemWash the column a l t e rn at e ly with acetone, wat e r, acetone at low fl ow rates, about 0.2 mL/min. Figure 1. Typical chromatograms of soybean phospholipids. Crude soybean lecithinDeoiled soybean lecithin CALCULATIONS 1 . Read from the calibration curves the amount of phos- pholipids in the injected volume of the solution of the lecithin sample (Pro c e d u re, 3). Typical ch ro m at ogra m s a re shown in Fi g u re 1. 2 . The phospholipid content (P), ex p ressed as perc e n t (mass/mass), is determined by the fo rmu l a : A 1 0 0 P = m Wh e re A = amount of phospholipid in mg determ i n e d from the calibration curve (Calculations, 1). m = mass of sample in mg (Procedure, 4). PRECISION 1 . S t atistics re l ating to the determ i n ation of PA, PC, PE and PI are shown in Table 1. NOTES Caution n-Hexane is extremely flammable. Avoid static electricity. The TLV in air is 50 ppm. A fume hood should be used at all times when using n-hexane. NUMBERED NOTES 1 . D e c reasing sep a ration perfo rmance is indicated by a bad sep a ration of the PE/N-acyl-PE peak. The column should then be replaced to ensure good rep ro d u c i b i l i t y. Use of a column oven is recommended for temperat u re s t ability; temperat u re ch a n ges will affect analyte re t e n- tion times. 2 . In some lecithins a sedimentation (usually suspended) m ay fo rm after a certain solution time. It has no infl u- ence on the analysis, but will affect the column. C e n t ri f u ging is re c o m m e n d e d, fo l l owed by decanting or fi l t e ring through a 0.45-m fi l t e r. REFERENCES 1 . S t a n d a rd Methods for the Analysis of Oils, Fats and D e rivat ive s , I n t e rn ational Union of Pure and Ap p l i e d C h e m i s t ry, 7th edn., Black well Scientific Publ i c at i o n s , 1987, IUPAC Method 5.302. 2 . B e a re - R oge rs, J.L., A. Bonekamp-Nasner and A. D i e ffe n b a ch e r, P u re Appl. Chem. 64:447 (1992). OTHER REFERENCES I U PAC Commission on Oils, Fats and Derivat ive s , Wo rking Rep o rt (VI.3), Intern ational Union of Pure and Applied Chemistry, July 12, 1989, pp. A7, M1. Page 2 of 2 SAMPLING AND ANALYSIS OF LECITHIN Ja 7b-91 Determination of Lecithin Phospholipids by HPLC Table 1 Summary of statistics from IUPAC phospholipid collaborative study a SamplenbMeancSrCVrSRCVR PA11112.580.272.141.5512.28 21111.680.171.451.129.56 3104.680.214.580.9620.61 41012.180.282.342.5120.63 51012.090.272.232.5421.01 6117.180.172.370.618.46 PC11017.240.201.190.975.61 21019.010.492.601.125.89 31114.440.342.440.865.95 489.640.131.400.222.25 5119.330.414.350.667.04 61114.740.402.680.835.66 PE11114.040.171.180.745.29 21015.800.150.981.106.98 31110.240.232.290.888.55