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Quantitative Analysis of Phospholipids by High Performance Liquid Chromatography

Name: ¾Ñ²¹Ò àËÅèÒ侺ÙÅÂì

keyword: ¡ÒÃÇÔà¤ÃÒÐËì»ÃÔÁÒ³¿ÍÊ¿ÍÅÔ»Ô´

Abstract: Ãкºàͪ¾ÕáÍÅ«Õ ·Õè§èÒÂáÅФÇÒÁäÇÊÙ§ä´é¶Ù´¾Ñ²¹ÒÁÒãªé㹡ÒÃÇÔà¤ÃÒÐËìÊÒÿÍÊ¿Òä·´ìËÅÑ¡ã¹àÅ«Ô¸Ô¹¨Ò¡¶ÑèÇàËÅ×ͧâ´Âãªé¤ÍÅÑÁ¹ì 3.9 ÁÔÅÅÔàÁµÃ x 30 ૹµÔàÁµÃ ºÃèØÊÒÃÃͧÃѺäÁâ¤Ã¾ÍÃÒ«ÔÅ «Ö觷Ó˹éÒ·Õèà»ç¹ÇÑ®ÀÒ¤¹Ôè§ áÅÐÁÕÍЫÕâµä¹äµÃìÅ / àÁ·¸Ò¹ÍÅ /¹éÓ / ¡Ã´¿ÍÊ¿ÍÃÔ¡ (85%),100 : 1 : 1 : 1 .5 (â´Â»ÃÔÁÒµÃ) à»ç¹ÇÑ®ÀÒ¤äËÅ ÊÒÿÍÊ¿Òä·´ì áµèÅеÑǵÃǨÇÑ´·Õè 205 á¹¹â¹àÁµÃ ¿ÍÊ¿ÒµÔ´ÔÅÍÔ¹¹Í«Ô·ÍÅ, ¿ÍÊ¿ÒµÔ´ÔÅà«ÍÃÕ¹, ¿ÍÊ¿ÒµÔ´ÔÅàÍ·¸Ò¹ÍÅÒÁÕ¹ áÅÐ ¿ÍÊ¿ÒµÔ´ÔŤÍÅÕ¹ ¨Ð¶Ù¡«ÐÍÍ¡ÁÒ·Õè 4.15, 5.31 , 6.02 áÅÐ 8.85 ÁÔÅÅÔÅԵà µÒÁÅӴѺ ÊÀÒÇдѧ¡ÅèÒǹÕéÊÒÁÒöá¡¿ÍÊ¿Òä·´ìËÅÑ¡¤×Í¿ÍÊ¿ÒµÔ´ÔÅÍÔ¹¹Í«Ô·ÍÅ ,¿ÍÊ¿ÒµÔ´ÔÅàÍ·¸Ò¹ÍÅÒÁÕ¹ áÅÐ ¿ÍÊ¿ÒµÔ´ÔŤÍÅÕ¹ ÍÍ¡¨Ò¡¡Ñ¹ä´éÍÂèÒ§ÊÁºÙóì ÊÒÁÒö»ÃÐËÂÑ´µÑÇ·ÓÅÐÅÒÂä´é»ÃÐÁÒ³ÃéÍÂÅÐ 70 áÅÐÂè¹ÃÐÂÐàÇÅÒ㹡ÒÃÇÔà¤ÃÒÐËìŧÍÂèÒ§ÁÒ¡ ÍÕ¡·Ñé§ãªéÊÒõÑÇÍÂèҧ㹡ÒÃÇÔà¤ÃÒÐËìµèÓ¶Ö§ 25 ṹ⹡ÃÑÁ «Öè§à»ç¹»ÃÔÁÒ³·ÕèµèÓ¡ÇèҢͧ Hurst áÅÐ Martin, JR.(100 ṹ⹡ÃÑÁ) ÊÀÒÇдѧ¡ÅèÒǹÕéËÒ»ÃÔÁÒ³¿ÍÊ¿Òä·´ìËÅѡ㹡ÑÁ¶ÑèÇàËÅ×ͧ¾ºÇèÒ ¢Í§ ºÃÔÉÑ·¸¹Ò¡Ã¼ÅÔµÀѳ±ì¹éÓÁѹ¾×ª¨Ó¡Ñ´ ÁÕÃéÍÂÅÐ 23.13 »ÃСͺ´éÇ ¿ÍÊ¿ÒµÔ´ÔŤÍÅÕ¹ (54.9%) , ¿ÍÊ¿ÒµÔ´ÔÅàÍ·¸Ò¹ÍÅÒÁÕ¹ (26.9%) , ¿ÍʿҵʹÔÅÍÔ¹¹Í«Ô·ÍÅ (18.2%) áÅТͧºÃÔÉÑ·ÍصÊÒË¡ÃÃÁÇÔÇѲ¹ì¨Ó¡Ñ´ ÁÕÃéÍÂÅÐ 5.67 »ÃСͺ´éÇ¿ÍÊ¿ÒµÔ´ÔŤÍÅÕ¹ (41.1%), ¿ÍÊ¿ÒµÔ´ÔÅàÍ·¸Ò¹ÍÅÒÁÕ¹ (37.7%) , ¿ÍÊ¿ÒµÔ´ÔÅÍÔ¹¹Í«Ô·ÍÅ (21.2%) àÍ·¸Ò¹ÍÅà»ç¹µÑÇ·ÓÅÐÅÒ·Õè´Õ㹡ÒÃÊ¡Ñ´¿ÍÊ¿ÒµÔ´ÔŤÍÅÕ¹¨Ò¡¡ÑÁ¶ÑèÇàËÅ×ͧ¢Í§ºÃÔÉÑ·ÍصÊÒË¡ÃÃÁÇÔÇѲ¹ì¨Ó¡Ñ´ ¿ÍÊ¿Òä·´ì·ÕèÊ¡Ñ´´éÇÂàÍ·¸Ò¹ÍÅ 20 »ÃÔÁҵà ÁÕÃéÍÂÅÐ 2.17 áºè§à»ç¹ 64.9 % ¿ÍÊ¿ÒµÔ´ÔŤÍÅÕ¹ , 29% ¿ÍÊ¿ÒµÔ´ÔÅàÍ·¸Ò¹ÍÅÒÁÕ¹ áÅÐ 5.9 % ¿ÍÊ¿ÒµÔ´ÔÅÍÔ¹¹Í«Ô·ÍÅ ¡ÒÃÊ¡Ñ´¿ÍÊ¿ÒµÔ´ÔŤÍÅÕ¹´éÇÂàÁ·¸Ò¹ÍŨдաÇèÒàÍ·¸Ò¹ÍÅ áµè¿ÍÊ¿Òä·´ì·Ñé§ËÁ´·ÕèÊ¡Ñ´ä´é¨Ð¹éÍ¡ÇèÒ ¿ÍÊ¿Òä·´ì·ÕèÊ¡Ñ´¨Ò¡¡ÑÁ´éÇÂàÁ·¸Ò¹ÍÅ 20 »ÃÔÁҵà ÁÕà¾Õ§ÃéÍÂÅÐ 1.22 áºè§à»ç¹ 69.7 % ¿ÍÊ¿ÒµÔ´ÔŤÍÅÕ¹, 27% ¿ÍÊ¿ÒµÔ´ÔÅàÍ·¸Ò¹ÍÅÒÁÕ¹ áÅÐ 3.3 % ¿ÍÊ¿ÒµÔ´ÔÅÍÔ¹¹Í«Ô·ÍÅ ¤ÇÒÁ¾ÂÒÂÒÁ㹡ÒÃà¾ÔèÁ sensitivity ËÃ×ÍÅ´¤èÒµèÓÊØ´¢Í§¡ÒõÃǨÇÑ´¿ÍÊ¿ÍÅÔ»Ô´·Õè¶Ù¡ªÐ´éÇÂàͪ¾ÕáÍÅ«Õ äÁè»ÃÐʺ¤ÇÒÁÊÓàÃç¨ à¹×èͧ¨Ò¡¿ÍÊ¿ÍÅÔ»Ô´äÁèÁռŵèÍ¡Òôٴ¡Å×¹áʧ¢Í§àÁ·¸ÔÅÕ¹ºÅÙ·Õè¤ÇÒÁÂÒǤÅ×è¹ 254, 490 áÅÐ 660 á¹¹â¹àÁµÃ áÅÐäÁèÊÒÁÒöŴËÃ×Íà¾ÔèÁ¿ÅÙàêà«ç¹« ´éÇÂâôÒÁÕ¹ 6 ¨Õ ä´é ¿ÍÊ¿Òä·´ìàÁ×èÍÅÐÅÒÂ㹤ÅÍâÿÍÃìÁ¨ÐÁÕ¤ÇÒÁÍÂÙèµÑÇ㹢Ǵá¡éÇàÅç¡·Õè»Ô´Ê¹Ô·äÁèÊÒÁÒöµÃǨÇÑ´¨Ó¹Ç¹·ÕèËÒÂ仢ͧ¿ÍÊ¿Òä·´ì ã¹ÃÐÂÐàÇÅÒ·ÕèÈÖ¡ÉÒ

Abstract: A simple and sensitive high pressure liquid chromatographic method was developed for the analysis of major phosphatides in soybean lecithin. A 3.9 rom. x 30 ern. column packed with u -porasil as stationary phase was used, using CH3 CN/CH3 OH/H2 O/85 % H3 PO4, 100 : 1:1 : 1.5 (v/v) as mobile phase. Individual phosphatide was detected at 205 nrn. Phosphatidylinositol, phosphatidylserine, phosphatidylethanolarnine and phosphatidylcholine was eluted at 4.15, 5.31, 6.02 and 8.85 ml. respectively. This condition can completely separate the three major phosphatides:phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. It also saves the mobile phase up to about 70 %. In addition, analysis time is much shorter and it can detect sample as low as 25 ng which is lower than that of Hurst and Martin, JR. (100 ng) . By this method, it was found that soybean gum from Thanakorn Co. contained 23.13 % of phosphatides composing of phosphatidylcholine (54.9 %), phosphatidylethanolamine (26.9 %), phosphatidylinositol (18.2 %) and soybean gum from Usahagamweewat Co., contained 5.67 % , composing of phosphatidylcholine (41.1 %), phosphatidylethanolamine (37.7 %), phosphatidylinositol (21.2 %). Ethanol was the best solvent for selective extraction of phosphatidylcholine in soybean gum from Usahagamweewat Co. About 2.17 % of phosphatides were extracted with 20 vol. ethanol and the extract comprised 64.9 % phosphatidylcholine, 29 % phosphatidylethanolarnine and 5.9 % phosphatidylinositol. Methanol is better than ethanol for selective phosphatidylcholine extraction but it is poorer in total phosphatides extraction. Only 1.22 % was extracted from the gum with 20 vol. methanol. The methanol extract contained 69.7 % phosphatidylcholine, 27 % phosphatidylethanolamine and 3.3 % phosphatidylinositol. The attempts to increase detection sensitivity or lower the minimum detectable amount of phospholipids eluted from high performance liquid chromatography (HPLC) were unsuccessful. Phospholipids did not affect methylene blue absorption at 254, 490 and 660 nm. They did not quench or enhance fluorescence of rhodamine 6 G. The phosphatides when dissolved in chloroform were relatively stable. In a tightly capped vial, there were no detectable amount degraded in one month at room temperature.

ÁËÒÇÔ·ÂÒÅÑÂà·¤â¹âÅÂÕ¾ÃШÍÁà¡ÅéÒ¸¹ºØÃÕ. ÊӹѡËÍÊÁØ´.

Address: ¡Ãا෾ÁËÒ¹¤Ã

Email: info.lib@mail.kmutt.ac.th

Name: ¤³Ôµ ¡Äɳѧ¡ÙÃ

Role: ÍÒ¨ÒÃÂì·Õè»ÃÖ¡ÉÒ

Created: 2553-06-25

Modified: 2553-06-25

Issued: 2553-06-25

ÇÔ·ÂÒ¹Ô¾¹¸ì/Thesis

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CallNumber: BIT88

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DegreeName: ÇÔ·ÂÒÈÒʵÃÁËҺѳ±Ôµ

Level: »ÃÔ­­Òâ·

Descipline: à·¤â¹âÅÂÕªÕÇÀÒ¾

Grantor: ÁËÒÇÔ·ÂÒÅÑÂà·¤â¹âÅÂÕ¾ÃШÍÁà¡ÅéÒ¸¹ºØÃÕ

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