HPLC Solvents, Acetonitrile and Methanol, Key Differences and Properties
HPLC溶劑乙腈和甲醇的關鍵區別和性能
Widely used in RP HPLC method development, Acetonitrile (ACN) and Methanol (MeOH) are the two most common solvents you will use to develop methods. So, besides the fact that Acetonitrile is well know to have a higher elution capacity than Methanol, what other properties should chromatographer's be aware of? Let's discuss a few that all chromatographers should know.
乙腈(ACN)和甲醇(MeOH)是在反向色譜柱方法開發中廣泛使用的兩種常見溶劑。所以，除了知道乙腈比甲醇有更高的洗脫能力這一事實外，色譜分析人員還應該知道其他的特性嗎?讓我們來討論一些所有色譜專家都應該知道的問題。
1 PREPARATIONS of MIXTURES (A/B)
混合物的製備(A/B)
First, a few comments about the preparation of mobile phase solutions.
首先，對流動相溶液的準備提出幾點意見。
Only the purely aqueous portion can be correctly adjusted for pH. Do not try and measure or adjust the pH of the organic or organic mixture.
只有純水溶液部分才能正確調整pH值。不要嘗試測量或調整有機或有機混合物的pH值。
There are two common methods of preparing binary mixture, V/V, mobile phase solutions.
製備二元混合物的方法有兩種，即V/V流動相溶液。
Method #1 is to fill a volumetric flask with a specific volume of the "A" solution, then fill the flask up to the line with the "B" solution.
方法#1是用特定體積的「A」溶液填充一個量瓶，然後用「B」溶液將量瓶填滿。
Method #2 is to fill a graduated cylinder(or volumetric flask) with a specified amount of "A" solution; fill a second graduated cylinder (or volumetric flask) with a specified amount of the "B" solution and then mix the contents of both together.
方法#2是用指定數量的「A」溶液填充量筒(或容量瓶)；用指定數量的「B」溶液填充第二個量筒(或容量瓶)，然後將兩者的內容混合在一起。
Whichever method you use, please fully document it in your HPLC method so anyone reading it will be able to accurately reproduce it. The two methods described above are both correct in design, but will result in solutions with different properties.
無論您使用哪種方法，請在您的高效液相色譜法中完整地記錄它，以便任何閱讀它的人都能準確地複製它。上面描述的兩種方法在設計上都是正確的，但是會產生不同性質的結果。
2 ABSORBANCE of UV LIGHT
紫外線吸光度
For HPLC grade solvent (*we should always use HPLC grade solutions in HPLC analysis) ACN has the lowest absorbance (~ 190nm) of the two making it well suited for low UV analysis. MeOH has a higher UV cut-off around 205-210 nm, slightly limiting its use in the very low UV ranges.
對於HPLC級溶劑(*我們在HPLC分析中應始終使用HPLC級溶液)，乙腈的吸光度(~ 190nm)在這兩種溶劑中最低，非常適合低紫外光分析。甲醇在205-210nm左右有較高的紫外光截止值，在非常低的紫外光範圍內略有限制。
3 SOLVENT SOLUBILITY
溶劑溶解性
There is a significant difference between ACN and MeOH in their ability to dissolve many types of buffer salts and samples. These differences are critical in method development.
乙腈和甲醇在溶解多種緩衝鹽和樣品的能力上存在顯著差異。這些差異在方法開發中至關重要。
3.1 Solubility of the Mobile Phase
流動相溶解度
A common reason for gradient runs to show poor reproducibility or to fail can be associated with running high concentrations of buffer with high concentrations of organic solution. While aqueous / organic solutions containing salt solutions of less than 10 mM concentration are not likely to precipitate under most gradient conditions (running to a max of 98% organic, not 100%), most buffer solution used with HPLC applications will have higher salt concentrations which may precipitate out of solution (resulting in clogs, leaks, plugs and in accurate results) when the analysis conditions contains high percentages of organic solvent. Be cautious when selecting organic composition values in RP methods. Make sure the solutions used will be stable under all concentrations used. Also verify that the buffering capacity is still present when high organic concentrations are used (as your buffer is diluted).
梯度運行顯示低重現性或失敗的一個常見原因可能與運行高濃度緩衝液和高濃度有機溶液有關。而含有濃度小於10mM鹽溶液的水溶液/有機溶液在大多數梯度條件下不太可能沉澱（最多98%是有機溶劑，而不是100%)，大多數與高效液相色譜應用一起使用的緩衝溶液會有更高的鹽濃度，當分析條件中有機溶劑含量較高時可能會從溶液中析出（導致堵塞，洩漏，插頭和不準確的結果）。在反向色譜法中選擇有機組成時要謹慎。確保使用的溶液在所有濃度下都是穩定的。還要驗證緩衝能力是否仍然存在，當使用高有機濃度時(當緩衝液被稀釋時)。
*Not sure if the salt will stay in solution?Just mix up a sample at the same concentration for a test. Look at it. Is there any turbidity or particulate visible? You have your answer.
不確定鹽是否會溶解?只要把同樣濃度的溶劑混合起來做測試就行了。觀察它，有任何渾濁或可見顆粒嗎?你就可以得到你需要的答案。
Methanol's overall better solubility characteristics(better than ACN) mean that it does a better job of dissolving most salts (esp NH4, K and Na) at higher concentrations resulting in better performance and less precipitation.
甲醇總體上具有更好的溶解度特性(優於乙腈)，這意味著它在較高濃度下能更好地溶解大多數鹽(尤其是NH4, K和Na)，從而獲得更好的性能和更少的沉澱。
3.2 Solubility of the Samples (effecton Peak Shape & Retention)
樣品的溶解度(對峰形和保留的影響)
A fundamental requirement of liquid chromatography is that the sample fully dissolves in the mobile phase (initial mobile phase). Dissolve the sample in the mobile phase or in a slightly weaker strength solution (not a stronger solution) before analysis. This insures it will be loaded onto the head of the column as a concentrated slug improving peak shape and RSD. If the sample doesnot fully dissolve in the mobile phase then you are not in fact analyzing the whole sample. Another area where Methanol may be superior to ACN can be found in its ability to fully dissolve more types of samples. This improved solubility may result in better overall peak shape. Methanol also has different selectivity than ACN (not just the elution strength) which may result in peaks eluting at different retention times than expecting. This is another reason why we always try different mobile phase mixtures containing either ACN or MeOH when developing RP methods. Never assume that one solvent will be better thanthe other. Too many novice chromatographer's use only ACN as their main organic solvent for method development. Please don't make their mistake as such a strategy indicates a lack of practical experience and knowledge. You must first try them both separately (ACN & MeOH) to evaluate the results with your sample (best to start with comprehensive gradients at different pH values, as applicable). You will be rewarded for putting in the initial time to test both types of solutions as no simulator has yet been developed which can predict a truly accurate result with your own sample(s). You may be surprised to learn how many samples show better peak shape and performance using MeOH solutions. If no improvement is seen, well at least you now know this because you tried itand can move forward with confidence.
液相色譜的一個基本要求是樣品完全溶解在流動相(初始流動相)中。在分析前，將樣品溶解在流動相或強度稍弱的溶液（不是更強的溶液）。這確保它將作為一個集中的段塞加載到柱的頂部，以改善峰形和RSD。如果樣品沒有完全溶解在流動相，那麼你實際上並沒有分析整個樣品。甲醇優於乙腈的另一個方面是它能完全溶解更多類型的樣品。這一改進的溶解度可能導致更好的整體峰形。甲醇的選擇性也不同於乙腈(不僅僅是洗脫強度)，這可能導致峰洗脫時間與預期的保留時間不同。這也是為什麼在開發反向方法時，我們總是嘗試使用含有乙腈或甲醇的不同流動相混合物的另一個原因。永遠不要假設一種溶劑會比另一種溶劑更好。太多的色譜新手只使用乙腈作為他們方法開發的主要有機溶劑。請不要犯他們的錯誤，這樣的策略表明缺乏實踐經驗和知識。您必須首先分別嘗試它們（乙腈&甲醇）用你的樣品來評估結果（在適用的情況下最好從不同pH值的全面梯度開始）。如果您在最初的時間內測試了這兩種類型的溶劑，那麼您將獲得回報，因為還沒有開發出能夠使用您自己的樣品來預測真正準確的結果的模擬器。您可能會驚訝地發現，有多少樣品使用甲醇溶液顯示出更好的峰形和性能。如果沒有看到任何改善，至少你現在知道了，因為你已經嘗試過了，並且可以滿懷信心地前進。
4 BACKPRESSURE:
背壓
ACN is less viscous than MeOH so will usually result in lower column and system back-pressures overall. Mixtures of ACN and Water will also exhibit an endothermic reaction (cooling the solution) which can trap gas inside the solution. If you pre-mix your mobile phase, let it rest for several minutes after preparation.
乙腈的粘性比甲醇小，因此通常會導致整體柱壓和系統背壓降低。乙腈和水的混合物也會發生吸熱反應(冷卻溶液)，從而在溶液中捕獲氣體。如果你預先混合你的流動相，讓它靜置幾分鐘後在製備。
MeOH is more viscous than ACN alone. It also has an unusual property where a 50/50 mixture of MeOH and Water will result in a much higher system and column back pressure than either MeOH or Water alone will. The effect is very Gaussian with a peak pressure observed with a 50/50 mixture. An exothermic reaction also results from an initialmixture of the two solutions giving off some gas. When preparing solutions itis best to allow the solution to rest for a few minutes to out-gass beforetopping off or using in the HPLC system.
甲醇比乙腈更粘稠。它還有一個不尋常的特性，就是甲醇和水的50/50混合物會產生一個比甲醇或水更高的系統和柱背壓。其效果是非常高斯的，它的峰值壓力是由50/50的混合物所觀察到的。兩種溶液在開始混合釋放出一些氣體也會產生放熱反應。在製備溶液時，最好是讓溶液靜置幾分鐘，然後在高壓液相色譜系統中使用。
I hope that this short discussion about some of the differences between these two popular HPLC solvents will aid you in developing better quality HPLC and LC-MS methods.
我希望這篇關於這兩種常用高效液相色譜溶劑的差異的簡短討論將有助於您開發出更好的高效液相色譜和LC-MS方法。