1. Measuring the pH of the mobile phase after the organic has been added

在添加有机相后测量流动相的pH值

pH meters are calibrated to give the correctpH readback in aqueous solution – the buffers you verify this with are aqueous.If you measure the pH with the organic added, the pH will be different to thatof measuring before organic addition.

在水溶液中，pH值被校准以提供正确的pH值——你需要确认的缓冲液是水溶液。如果你用添加了有机相的水溶液来测量pH值，pH值就会与有机相添加前的pH值不同。

However, the most important point is to beconsistent.If you do always measure pH after theorganic is added, make sure you state this in the method so that everyone doesit the same way. It won’t be 100% accurate, but at least it will be consistent.This is probably more important than having the exact pH.

然而，最重要的一点是要保持一致。如果你总是在添加有机相后测量pH值，确保你在方法中说明了这一点，以便每个人都用同样的方法。它不会百分百准确，但至少会是一致的。这可能比精确的pH值更重要。

2. Not using a buffer

没有使用缓冲液

Buffers are present to control pH and resist a change in pH.Many other parts of method (e.g. sample matrix, CO2inair, source of water used for your mobile phase) can change the pH of themobile phase causing shifts in retention, peak shape and peak response.Formicacid, TFA etc. are not buffers.

缓冲液是用来控制pH值和抵抗pH值变化的。方法的许多其他部分(例如样品基质、空气中的二氧化碳、流动相中水的来源)可以改变流动相的pH值，从而导致保留、峰形和峰值响应的变化。甲酸，三氟乙酸等不属于缓冲试剂。

3. Not using the buffer in its correct pH range

没有在缓冲液正确的pH范围内使用

Each buffer salt has a 2 pH unit wide rangeover which it provides the optimal pH stability. Outside this window the saltis ineffective at resisting change in pH.Either use your buffer within thecorrect range or pick a buffer whose range covers the pH you require.

每个缓冲盐有2个pH值单位范围，它提供了最佳的pH稳定性。在缓冲范围外面，盐对pH值的变化是无效的。要么在正确的范围内使用缓冲区，要么选择一个范围覆盖所需pH值的缓冲区。

4. Adding buffer to organic

将缓冲液加入有机相中

Mixing aqueous buffer into the organic phasecarries a high risk of the buffer being precipitated – in many cases so finely that it may notbe obvious it has happened.Alwaysadd the organic to the aqueous phase, this greatly reducesthe risk of buffer precipitation.

将水的缓冲液混合到有机相中，会导致缓冲液沉淀的高风险——在许多情况下，变化微小，以至于可能不太明显。常常是将有机物质添加到水相，这大大降低了缓冲沉淀的风险。

5. Using the pump to mix gradients from 0%

使用泵的梯度混合从0%开始

Modern pumps are very effective at mixingmobile phases and degassing online, however not everyone who ends up using yourmethod has a high quality pump. Premix your A and B starting mix to a singlesolution that runs at 100% on line A. e.g.

现代泵在混合流动相和在线脱气上非常有效，然而，并不是每个使用你的方法的人都有一个高质量的泵。预混合你的A和B，混合成一个单一的溶液，在A管路上100%运行。

Prepare the starting mixture by mixing 950mlAqueous with 50ml organic, then filter and degas. This reduces variabilitybetween HPLCs, reduces the risk of bubbles and precipitation in the system.

将950mL的水溶液与50mL的有机溶液混合，然后过滤和脱气。这降低了HPLC之间的可变性，降低了系统中气泡和沉降的风险。

Note however that 95:5 mixed on the pumpwill not give the same retention time as 95:5 premixed in the bottle – younormally need to add a few more percent organic when premixing.

注意，在泵上混合的95:5不会和在瓶中预拌的95:5相同的保留时间——你通常需要在预混合时添加更多的有机成分。

6. Not using the correct pH modifying acid or base for your buffer

没有使用正确的酸或碱调节你的缓冲液的pH值

Only use the acid or base that forms thebuffer salt you are using. E.g. sodium phosphate buffers should be adjustedwith only phosphoric acid or sodium hydroxide.

只使用你正在使用的缓冲盐的酸或碱。举例来说，磷酸钠缓冲液应该仅用磷酸或氢氧化钠来调节。

7. Not stating the full information of your buffer in the method e.g.     weigh 5g of sodium phosphate into 1000ml of water

没有在方法中说明你的缓冲液的全部信息，例如，将5g的磷酸钠放入1000ml的水中

The type of buffer (mono, di or tribasic)determines its pH buffering range.

缓冲剂的类型(单盐基，二盐基，三盐基)决定了它的pH缓冲范围。

The required molarity is what determines the buffer strength.5g of anhydrous sodium phosphate and 5g of monohydrate sodium phosphate willhave different buffer strengths and will affect retention.

所需的质量浓度决定了缓冲强度。5g无水磷酸氢钠和5g一水合磷酸氢钠会有不同的缓冲强度，并会影响到保留。

8. Filling lines with organic without checking what was in there before

用有机相的填充管路，不检查之前的内容

If the previous method used buffer in line Band your method uses organic in line B there’s a good chance you willprecipitate buffer in your pump tubing / pump head. I did it in my early daysand it caused a lot of damage.If in doubt – flush it out(80:20water : organic).

如果前一种方法在B管路中使用了缓冲液，而你的方法在B管路中使用了有机相，那么缓冲液很有可能会在你的泵管路/泵头中沉淀。我在早期就这样做了，并且造成了很大的伤害。如果有怀疑——把它冲出来(用80:20水:有机相)。

9. Propping up bottles to get last drop out

一直到瓶子的最后一滴流动相被使用掉

It’s 5 to 5 and you’ve barely got enoughmobile phase to finish the run – it’ll be running on fumes by the last fewsamples. Apart from the risk orrunning your pump and column dry, mobile phases evaporate from the surface, sothe mobile phase at the top of the bottle will have changed composition fromthe bulk. This portion from the top is exactly what will be running through thecolumn if you use the last dregs in the bottle.

如果没有足够的流动相来完成运行——将有50%的可能性，气体将会在最后几个样品的中运行。除了磨损你的泵和 走干的风险，流动相从表面蒸发，所以在瓶子顶部的流动相与大多数体积相比成分将会改变。如果你使用瓶子里的最后少量的流动相，顶部的这个部分恰好是贯穿柱子的东西。

10. Using sonication to degas mobile phase

使用超声仪对流动相进行脱气

It’s great for making sure all your buffersalts have dissolved, but it’s the least effective method of degassing and itquickly heats up the mobile phase causing the organic portion to evaporate.Save yourself problems later –take 5mins to vacuum filter your mobilephase– it degasses and filters in a single step.

这对确保你所有的缓冲盐溶解都很好，但这是最不有效的脱气方法，它会迅速加热流动相，导致有机部分蒸发。节省你日后的问题——用5分钟的时间来过滤你的流动相——它会在一个步骤中脱气和过滤。下图为各种脱气的效率示意图！

微信图片_20210623001515

（文章来源：分析圈     由小析姐整理编辑）