主题：【在线讲座33期】液相色谱柱使用疑难问题解析(答疑结束）

原文由 lijin79833313(lijin79833313) 发表:
plexu您好！我有以下问题想请教，
请问：普通的C18柱能作为正相色谱柱使用吗？正相色谱体系中最常用也最普通的是那种色谱柱。正相柱在使用过程中与反向柱相比有什么需要注意的地方，谢谢！！

原文由 plexu(plexu) 发表:

原文由 小卢(luxw) 发表:
plexu您好！我有以下问题想请教：
柱子在什么情况下可以清洗一下筛板呢？
原来也讨论过这个问题，我也拆下来清洗过，但我看到柱前段的污染更甚，于是就用刀片刮了刮，然后把清洗好的筛板安装上去。问题解决了，但使用寿命会不会减少呢？

一般不建议把筛板拆下来清洗，也不建议挖补已污染的柱头填料，因为这样会影响柱床的稳定性，修补后的色谱柱的性能并没有很好的保障。用强溶剂反冲清洗色谱柱是色谱柱污染后可以采取的措施，不过不要等污染已经很严重才想起这样做，定期用甲醇进行清洗维护更好些。反冲清洗再生的方法用了不奏效，一般也可以把色谱柱报废了，实在舍不得，拆筛板挖填料也算是最后的手段。

原文由 毛毛儿(yangxy12345) 发表:
plexu您好！我有以下问题想请教:
1、色谱柱的柱头类型与不锈钢毛细管接头有6种连接方式（在讲义里面提到），那么我们用户一般是液相色谱仪是固定某一个厂家，而是根据样品检测需要更换不同类型的色谱柱，那么怎么判断我所购买的色谱柱是否与不锈钢毛细管是否匹配呢，我之前只是知道安捷伦和waters都有规定他们自己家的柱子与自己家的仪器配套是最合适的，而其他厂家的色谱柱都很少提及，在不知道的情况下，我们该怎么选择？月旭的色谱柱柱头类型属于哪一种类型呢？

原文由 plexu(plexu) 发表:

原文由 小卢(luxw) 发表:

原文由 plexu(plexu) 发表:

原文由 小卢(luxw) 发表:
plexu您好！我有以下问题想请教：
第三个问题：
预柱或保护柱用还是不用的问题！
原来分析中药品种时，我一直都是用保护柱。但来到新公司后，发现大家都没有使用，几个实验室连保护柱都没找到一个，也就是说大家从来都没有用过。后来问一个老员工，说是有可能影响药品分析。
我就想问：安装保护柱后会影响样品分析吗？我们做的大多是头孢类的抗生素。

如果用一个质量好的又和分析柱匹配保护柱，应该不会影响分析。匹配指的选用柱芯填料和分析柱填料在粒径和固定相类型上一致，质量好，首先要求柱芯装填质量一定要好，不能因为是柱芯，就随便把填料用干法往里面一放就完事了；另外柱芯卡套接入系统的产生的死体积要小。
一个质量好的保护柱，有时还能增加分离柱效。

这个问题我考虑过！但有个担心的问题，就是由于我们的大多检测标准时欧盟或美国药典标准，因此填料、柱长、直径都固定了，如果选择保护柱的话，还需要考虑那么多吗？

美国药典欧洲药典对填料和柱长并没有规定很死，是允许在一定范围内调整的，加保护柱肯定不违反药典的规定。

原文由 yongzhbenq(yongzhbenq) 发表:

原文由 plexu(plexu) 发表:

原文由 小卢(luxw) 发表:
这个问题我考虑过！但有个担心的问题，就是由于我们的大多检测标准时欧盟或美国药典标准，因此填料、柱长、直径都固定了，如果选择保护柱的话，还需要考虑那么多吗？

美国药典欧洲药典对填料和柱长并没有规定很死，是允许在一定范围内调整的，加保护柱肯定不违反药典的规定。

前面讲了一些欧洲药典的规定，后面可以再看看美国药典的规定

Adjustments to the Chromatographic System

As mentioned, <621> allows a number of adjustments to the chromatographic system in response to issues with a procedure's system suitability. These are the maximum allowable variations unless the individual monograph directs otherwise.

Ratio of components in mobile phase: Although the replacement of any solvent in the mobile phase constitutes a change and not an adjustment, the amounts of the minor components (specified as 50% or less) in the mobile phase can be adjusted up to ±30% relative to the particular component. However, the change in any component cannot exceed ±10% absolute (that is, in relation to the total mobile phase). Adjustments can be made to one minor component in a ternary mixture.

pH of mobile phase: The pH of the aqueous buffer used in the preparation of the mobile phase can be adjusted to within ±0.2 units of the value or range specified.

Column temperature: The column temperature can be adjusted by as much as ±10 °C.

Concentration of salts in buffer: The concentration of the salts used in the preparation of the aqueous buffer for the mobile phase can be adjusted up to ±10% relative to the particular component, provided the permitted pH variation (see above) is met.

Wavelength of UV–vis detector: Deviations from the wavelengths specified in the method are not permitted.

Stationary phase:

• Column length: The column length can be adjusted by as much as ±70%.
• Column inner diameter: The column inner diameter can be adjusted, provided that the linear velocity is kept constant (see Flow Rate).

Flow rate: When column dimensions have been modified, the flow rate can be adjusted using the following formula:

where

F₁ is the flow rate indicated in the monograph, in milliliters per minute;

F₂ is the adjusted flow rate, in milliliters per minute;

l₁ is the length of the column indicated in the monograph;

l₂ is the length of the column used;

d₁ is the column inner diameter indicated in the monograph; and

d₂ is the internal diameter of the column used.

Additionally, the flow rate can be adjusted by ±50% (2).

Injection volume: The injection volume can be reduced as far as is consistent with accepted precision and detection limits, but no increase is permitted.

Particle size: Particle size can be reduced by as much as 50% but cannot be increased.

Column Dimensions: Inner Diameter, Length

The chromatographic purity test included in the hydroxyzine hydrochloride monograph (8) required the use of two 10 cm × 3 mm columns coupled in series and containing packing L3 with a flow rate of 0.4 mL/min. It would be advantageous to use one column with standard dimensions of 4.6 mm i.d. and a 25-cm length. Chapter <621> allows the simultaneous change of column inner diameter and length and recommends the flow rate be adjusted according to equation 1 given earlier.

The new flow rate calculated using the formula is 1.2 mL/min. When a flow rate of 1.0 mL/min was used with a 4.6-mm i.d. and 25-cm length column, the observed resolution was greater than 3. The current official monograph includes the revised column dimensions of 4.6-mm i.d. and 25-cm length and flow rate of 1.0 mL/min