主题:【讨论】与HPLC色谱柱有关的十大神话

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plexu
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似是而非,就是这十种说法初看起来有道理,但其实不对或者不完全对的意思。应楼上网友的要求,我将在本贴中,逐条把有关这十种说法的来龙去脉翻译过来。
plexu
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Myth 1: You Can't Reverse an HPLC Column
False. In practice, a high performance liquid chromatography (HPLC) column is packed at pressures much higher (often as much as twofold) than its maximum operating pressure. Thus, if the proper slurry solvent was used and the time allotted for the packed bed to be stabilized, a well-packed column should be able to work in both directions. Some reasons why one would want to flow a column in the opposite direction include backflushing operations in column switching, rinsing a column with stubborn strongly sorbed sample at its entrance (a shorter path length than to flush the insoluble materials through the entire column length), and flushing trapped particulates to decrease pressure buildup.
There is one exception in reversing the flow of an HPLC column. If the manufacturer has used a higher porosity frit at the entrance to the column, by reversing the column it might be possible to flush particles out of the packed bed. When the column is packed at the factory, the porosity of frit at the outlet of the column must be lower than the smallest particle size of the column. For example, if the column packing has an average diameter of 5 μm and a particle size distribution of 3–7 μm, the frit used at the column exit must be less than 3 μm so that there is no possibility that particles will escape the packed bed when the column is being used. For that case, most manufacturers choose a 2-μm frit.
Why would a manufacturer have different porosity frits at the column inlets and outlets? Simple. A higher porosity frit has fewer tendencies to plug than a lower porosity frit. A 0.5-μm frit will plug faster than a 2-μm frit. So, to prevent rapid pressure buildup and customer complaints, a manufacturer might use a more forgiving, larger porosity frit at the inlet. Usually, the column will be marked with an arrow that indicates that it should be used in one direction only. Before reversing an HPLC column, it is best to consult the column instruction sheet or check with the column manufacturer to see if the column can be reversed.
plexu
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神话一: HPLC色谱柱不能反转使用

错误!实际上HPLC色谱柱的填装压力比最大使用压力高很多(通常会高2倍)。如果装柱时使用了恰当的匀浆剂,并且分配一定的时间使柱床稳定,一支填装良好的色谱柱是完全可以双向使用的。需要反向使用色谱柱的情况包括:柱转换时的反冲、柱头被强保留物质吸附污染的色谱柱清洗(反冲的冲洗路径更短更合理)、冲洗残留在筛板上的颗粒物以降低柱压或防止柱压升高。
反向使用色谱柱有一个例外,就是生产商在色谱柱的进样端使用了孔径更大筛板的情况, 反向使用可能会将填料从柱床冲出。色谱柱在工厂填装时,出口端的筛板孔径必须比色谱柱中最小颗粒的粒径还要小。譬如,色谱填料平均粒径是5μm,粒径分布范围是3–7 μm, 出口端筛板孔径必须小于3μm ,使填料没有可能从柱床跑到色谱柱筛板外面。大多数生产厂家选择的筛板孔径是2-μm。                                   
为什么有些色谱柱生产厂家会选择入口端和出口端采用不同孔径的筛板呢?很简单,孔径大的筛板相对孔径小的筛板不容易堵,一个 0.5-μm的筛板会比一个 2-μm的筛板被堵得快。所以为了避免柱压上升过快和客户的抱怨,生产商会折中地选择在入口端用一个孔径稍大的筛板,通常色谱柱上就会标一个箭头表示只能在一个方向使用。在反向使用 HPLC色谱柱时,最好仔细看一下说明书或者咨询一下色谱柱生产厂家是否能反向使用。
plexu
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原文由 ecco2(ecco2) 发表:
附件下载下来后打不开好像是损坏的文档


文档没有损坏,也能打开的,你再试试看。
lr2008_ongi
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神贴,建议楼主继续翻译,英语底子不好啊
plexu
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Myth 2: All C18 (L1) Columns Are the Same
False. In the early days of HPLC, octadecylsilane (most often referred to as an ODS or C18 phase) was one of the first bonded stationary phases that became available for the new technique called "reversed-phase chromatography." It became the standard phase for reversed-phase chromatography and quickly was adopted by most practitioners. Because the pharmaceutical industry was an early adopter of HPLC and regulatory bodies did not want to bless a particular manufacturer''s brand of column, the FDA and USP developed a classification system that gave a generic designation for each new method that was submitted under a new drug application. For HPLC columns, an "L" designation was given and because C18 was used in a majority of the submittals, it became "L1." As additional phases were added, they were given their own "L" number (for example, L7 C8, L10 CN, L11 Phenyl, and so forth). Unfortunately, this designation system proved to be unreliable because each commercial C18 column was synthesized differently using silica gel as the base material and the resulting reversed-phase columns had different properties. For example, some manufacturers used octa-decylmonochlorosilane and a low surface area silica gel (Figure 1) while others used the same silane but bonded it to a higher surface area silica gel. These two C18 columns would behave differently with the latter having more C18 phase than the former. A lower coverage bonded phase sometimes had unreacted silanols that caused mixed retention mechanisms. Some manufacturers use di- and trichlorosilanes and polymerized the bonded phase to form a thicker layer with different diffusional properties. To cut down on unreacted silanols that remained after the bonding process, some manufacturers endcap these silanols with a small silane (for example, trimethylmonochlorosilane). Silanols are sometimes responsible for tailing of basic compounds at intermediate pH values. Some manufacturers even went further and double-endcapped with a second small silane to provide a more inert surface. Some manufacturers used polymeric base materials and then bonded C18 moieties to their surface resulting in a totally different C18 packing material, yet it was still considered an "L1." Others use organoalkoxysilanes that display a different degree of reactivity and can produce a slightly different C18 bonded phase than would an organochlorosilane reagent.
yongzhbenq
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接上面的,我来顺带帖个图片解释一下
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Last edit by yongzhbenq
plexu
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神话二:所有的C18(L1)柱是一样的。

错误! 在HPLC发展初期,十八烷基硅烷(常被称为ODS或C18)是最早的键合固定相之一,也导致了“反相色谱”这个新技术的诞生。C18 成为了反相色谱的标准固定相并被大部分色谱工作者所采用。制药工业是HPLC技术的最早使用者,监管部门不想偏袒任何一家色谱柱生产厂商的品牌名字,FDA和USP编写了一套分类体系,给每一种药物应用方面的新方法一个通用名称。对HPLC色谱柱,命名为“L”,C18因为出现在大部分送审材料中,理所当然就成为了“L1”。随着另外固定相的增加,分别都有了自己的“L”序列号(如:L7是C8,L10是CN基,L11是苯基等等)。
很不幸,这个命名体系被证明是不可靠的。因为每种商品化的C18色谱柱,虽然同样是选用硅胶作为基体,但各自都有自己特定的填料键合合成工艺,因此色谱性能也不相同。譬如,有的生产厂商采用十八烷基一氯硅烷键合试剂和低表面积的硅胶(见图一所示),而其它一些厂家采用同一硅烷试剂但选用了表面积更高的硅胶基体。这两种C18柱表现的色谱性能不同,后者C18固定相的键合比例大于前者。较低的固定相键合比率,表面未反应的硅醇基较多,有时混合保留机理起主要作用。某些色谱柱生产商使用二氯硅烷和三氯硅烷作键合试剂,将键合相聚合反应形成一很厚的具有不同扩散特性的疏水层。为使键合后硅胶表面未反应的硅醇基比例降低,有些生产商用小分子的硅烷试剂(如三甲基氯硅烷)封尾。硅醇基是在中性条件下测定碱性物质时导致色谱峰拖尾的原因之一,有些生产厂家,更进一步的做法是采用第二种小分子硅烷进行双封尾工艺,以提供一个更加惰性(疏水性)的硅胶表面。另外有些生产商采用聚合物基体材料进行C18 键合,生产出一种完全不同的C18填料,但仍然被归类到“L1”。还有厂家采用反应性能不同的硅烷化试剂organoalkoxysilanes,制造出一种与氯硅烷反应产生的不同的C18 固定相。
土老冒豆豆
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翻译成中文好看多了,还真是长知识。
现在用的柱子一般都会标有流动相方向的,但我用过一款默克公司的柱子,没有明确的箭头指示流动相方向的,是不是意味着这款柱子可以双向使用啊。
plexu
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原文由 土老冒豆豆(土老冒豆豆) 发表:
翻译成中文好看多了,还真是长知识。
现在用的柱子一般都会标有流动相方向的,但我用过一款默克公司的柱子,没有明确的箭头指示流动相方向的,是不是意味着这款柱子可以双向使用啊。


月旭的色谱柱产品两端的筛板孔径是一样的,尽管柱身标签上也有箭头指示流动相方向,但仍然可以双向使用,箭头的方向只是说明大部分情况下用这个方向比较好。碰到需要清洗强保留物质和颗粒物时,月旭的柱子可以放心地进行反向使用,建议大家每隔一段时间将柱子进行反冲清洗,以避免颗粒物累积导致柱压升高。长时间不反冲色谱柱,会导致颗粒物不可逆地粘附在筛板上。
具体默克的柱子是否可以双向使用,还是咨询一下厂家比较稳妥。