The mobile phase is equivalent to the liquid phase of blood , and there are various things to pay attention to during use. Among them, there are some “pitfalls” that must be paid attention to.
01. Measure the pH of the mobile phase after adding organic solvent
If you measure pH with an organic additive, the pH you get will be different than before adding the organic solvent. However, the most important thing is to be consistent. If you always measure pH after adding the organic solvent, make sure to state your steps in the method you use so that others will follow the same method. This method is not 100% accurate, but at least it will keep the method consistent. This may be more important than getting an accurate pH value.
02. No buffer used
The purpose of a buffer is to control the pH and prevent it from changing. Many other methods change the pH of the mobile phase, which can cause shifts in retention time, peak shape, and peak response.
Formic acid, TFA, etc. are not buffers
03. Not using buffer within the normal pH range
Each buffer has a 2 pH unit range width, within which it provides the best pH stability. Buffers outside this window will not provide effective resistance to pH changes. Either use a buffer in the correct range, or choose a buffer that covers the pH range you need.
04. Add buffer to organic solution
Mixing a buffer solution with an organic phase will most likely cause the buffer to precipitate. In many cases, even if precipitation has occurred, it is still difficult to detect. Remember to always add the organic solution to the aqueous phase, which can greatly reduce the chance of buffer precipitation.
05. Mix the concentration gradient from 0% with a pump
Pumps available today can effectively mix mobile phases and degas inline, but not everyone using your method will have a high quality pump. Mix A and B into a single solution and run it 100% inline.
For example, 950 ml of organic starting mixture can be prepared by mixing with 50 ml of water. The advantage of this is that it can reduce the variability between HPLCs and reduce the possibility of bubbles and precipitation in the system. It is worth noting that the ratio of the pump mixture is 95:5, which does not mean that the pre-mixed retention time in the bottle is also 95:5.
06. Not using the correct modified acid (base) to change the buffer
Only use the acid or base that forms the buffer salt you are using. For example, a sodium phosphate buffer should be prepared with only phosphoric acid or sodium hydroxide.
07. Not stating all the information about the buffer in the method, such as adding 5g of sodium phosphate to 1000ml of water .
The type of buffer determines the pH range that can be buffered. The required concentration determines the buffer strength. 5 grams or anhydrous sodium phosphate and 5 grams of monosodium phosphate monohydrate have different buffer strengths.
08. Adding organic solvents before checking
If the previous method used a buffer solution for baseline B, and your method uses an organic solution for baseline B, you can hopefully settle the buffer in the pump tubing and pump head.
09. Lift the bottle and empty the last drop
There is a good chance that you will not have enough mobile phase to complete the entire run and your sample will smoke. Besides the possibility of burning out the pump system and column, the mobile phase will evaporate completely and the mobile phase at the top of the bottle will change.
10. Use ultrasonic degassing mobile phase
The most important point is to make sure all the buffer salts are dissolved, but this is the worst way to degas and will quickly heat up the mobile phase, causing the organic components to evaporate. To save unnecessary trouble later, take five minutes to vacuum filter your mobile phase.
Post time: Aug-27-2024