布莱代尔油脂萃取法

Procedure
Reagents
Methanol, absolute, analytical reagent: chloroform, analytical reagent.
Lipid extraction and purification
The following procedure applies to tissues like cod muscle that contain 80±1% water and about 1% lipid. Each 100g sample of the fresh or frozen tissue is homogenized in Waring Blendor for 2 minutes with a mixture of 100 ml chloroform and 200 ml methanol. To the mixture is then added 100ml chloroform and after blending for 30 seconds, 100 ml distilled water is added and blending continued for another 30 seconds. The homogenate is filtered through Whatman NO. 1 filter paper on a Coors NO. 3 Buchner funnel with slight suction. Filtration is normally quite rapid and when the residue becomes dry, pressure is applied with the bottom of the beaker to ensure maximum recovery of solvent. The filtrate is transferred to a 500-ml graduated cylinder, and, after allowing a few minutes for complete separation and clarification, the volume of the chloroform layer (at least 150 ml) is recorded and the alcoholic layer removed by aspiration. A small volume of the chloroform layer is also removed to ensure complete removed of the top layer. The chloroform layer contains the purified lipid.
For quantitative lipid extraction the lipid withheld in the tissue residue is recovered by blending the residue and filter paper with 100 ml chloroform. The mixture is filtered through the original Buchner funnel and the blender jar and residue are rinsed with a total of 50 ml chloroform. This filtrate is mixed with the original filtrate prior to removal of the alcoholic layer.
Adaptation to Other Materials
The above procedure can be applied directly to any 100-g sample containing 80g water. Many alterations of the procedure are permissible but it is imperative that the volumes of chloroform, methanol, and water, before and after dilution, be kept in the proportions 1:2:0.8 and 2:2:1.8, respectively. These ratios represent the total volumes present in the ternary systems, including the water present in the sample. Thus, in the extraction of materials that do not contain 80% water, either the size of the sample can be adjusted so that they contain 80-g water or 100g samples can be used and volumes of chloroform and methanol changed to give the correct proportions. In cases where the moisture content is much less than 80% (e g. Fish meal), it is necessary to add distilled water. When material containing a large amount of lipid is used, or where the supply of material is limited, the size of sample can be reduced and the above solvent quantities scaled down to meet requirements.
Determination of lipid content
A portion of the lipid extract containing 100-200mg lipid is evaporated to dryness in a tared flask and the weight of the lipid residue determined. Evaporation, facilitated by a stream of nitrogen, is carried out in a water bath at 40-50℃and the residue is dried over phosphoric anhydride in a vacuum desiccator. After weighing, a small volume of chloroform is added to each flask to detect the presence of non-lipid material (insoluble). If non-lipids are present, the chloroform is carefully decanted and the flask rinsed three times with chloroform. The dry weight of the residue is determined and subtracted from the initial weight. The lipid content of the sample is calculated as follows:
Total lipid=weight lipid in aliquot×volume of chloroform layer/volume of aliquot
试剂
甲醇、绝对、分析试剂:氯仿、分析试剂。

脂质提取和纯化
该检测程序适用于如鳕鱼肌肉组织，里面含有80±1%的水分,约1%的脂质。

将100克新鲜或冰冻的样品组织中加入100毫升氯仿和200毫升甲醇并在绞碎机绞碎两分钟。

然后在混合物里再加上100毫升氯仿混合30秒使其均匀,再加入100毫升蒸馏水混合30秒。

浆液用1号滤纸通过布氏漏斗缓慢过滤。

当渣变得干燥时过滤通常很迅速 ,烧杯底部压力应较大,以确保最大限度的回收的溶剂。

把滤液转移到一个500毫升量筒里面,并且在几分钟内使其完全分离和澄清、记录氯仿层的体积(至少150毫升)而酒精层被吸走。

少量的氯仿层也应该被移除,从而确保完全移除的上层酒精层。

被纯化的脂质溶解在氯仿层里面。

为提取残留在组织中少量的脂质，应该把残留物溶解在100ml氯仿中，然后用滤纸过滤。

混合物通过原来的布氏漏斗，搅拌罐和残渣用50毫升氯仿冲洗。

该滤液与原来除去酒精层的氯仿层混合。

适用于他材料
上述过程可直接用于任何含有80克水的100g样品。

很多对程序的改变是允许的,但是在稀释前后氯仿、甲醇和水的比例一定要控制在1:2:0.8与2:2:1.8之间。

这三种试剂的比例是代表三元系统的成分组成，还包括样品中表现出来的水分。

因此,在提取含水没有80%的原料时,或者把他样品做适当调整，以使他们含有80g水分，或者用就100g样品，而加入的氯仿和乙醇做适当的折算。

在某些情况下样品水分含量远低于80%(例如，鱼肉),必须加入蒸馏水。

当使用的原料含有大量的脂质时,或在原料的供应有限的情况下,样品的可以适当减小，而溶剂的用量按比例减小，以使其满足要求。

脂质含量测定
部分油脂萃取可以得到100 - 200毫克脂质，把它们放入配衡烧瓶中蒸干，脂类的残渣的重量是一定的。

残渣通过在40-50℃水浴蒸发，方便的话通入氮气流,残渣用磷酸酐在真空干燥器里面干燥。

在称量之后，在每个烧瓶中加入少量的氯仿以检验非脂质成分(难容的)。

如果存在非脂质,用氯仿小心地对烧瓶冲洗三次。

残留物干重的测定,并减去最初的重量。

样品脂质含量的计算方式如下:
总脂质=脂质在等分数重量×氯仿层体积/体积的等分数。