How to Prepare Mobile Phases - Solvent Mixture Ratios
Mobile Phase: 50 % aqueous ethanol solution
How would you prepare this solution?
Solvent mixtures are generally prepared in terms of volume ratios (v/v) or weight ratios (w/w). Since the volume of a solution varies with temperature, preparing solvent mixtures based on weight ratios provides better reproducibility, but doing so is more complicated, so it is probably safe to assume mixtures are normally prepared based on the volume ratios. However, in some special cases, such as when mixing highly viscous solutions like amines, weight-volume ratios (w/v) are sometimes used as well.
In addition, notation methods can vary widely for mobile phase conditions, depending on the reference document, HPLC database, or other source.
It is extremely rare to see mobile phase preparation procedures written in detail, such as "add 340 µL of phosphoric acid to 100 mL of water." Rather, notation such as "20 % aqueous acetonitrile solution" or "acetonitrile - water (40:60)" is used. Sometimes, notation methods that do not total 100 %, such as "acetonitrile/water = 21/5" or "methanol/water/phosphoric acid = 95/5/0.3" are used.
Though there is no fixed method for the notation of mobile phase composition, the notation method must allow preparing mobile phases in a manner that enables reproducing the analysis according to the given analytical conditions and it must be possible to understand the notation method correctly.
Meaning and Preparation Method of 50 % (v/v) Aqueous Ethanol Solution
Given the notation "ethanol/water = 1/1," the method indicated in Procedure 1 below is apparently widely used for preparation.
However, the same Procedure 1 is often used to prepare solutions indicated as "50 % aqueous ethanol solution." But, according to a dictionary of chemistry, Procedure 2 is actually the preparation method that uses correct volume percentages (see table). If so, the volume ratios indicated in terms of percent and ratios, such as "1:1," eventually result in different mobile phase composition. In other words, the density of solvent mixtures is not a simple mean value of the densities of the constituent solvents, so the two methods indicated above result in different mobile phase compositions. For example, at temperatures near room temperature (25 °C),mixing 50 mL water with 50 mL ethanol does not result in a volume of 100 mL, but rather it shrinks slightly to about 96 mL. In general practice, the easier Procedure 1 is more widely used. Therefore, it is recommended that notation should be in terms of a ratio such as "XXX/YYY = 2/3."
Temperature Effects on Solvent Volume
As indicated above, the density of solutions is affected by the ambient temperature. When a solvent solution is prepared immediately after taking the solvent from storage, its temperature can be much lower than the laboratory room temperature and mixtures involving methanol and water can be warm due to exothermic reaction. Therefore, to prepare mobile phases with good reproducibility, it is recommended that solvents be placed in a cold or hot water bath for a while before use, until they approach room temperature.
Mixing Solvents Using Two Pumps
Isocratic methods, often used with organic solvents and water in reverse phase chromatography, involve either using two pumps to deliver two types of mobile phase in a high pressure gradient system, then mixing them in a mixer or other closed system, or premixing the components in a bottle and using one pump for delivery. However, note that these two methods can result in different retention times, due to changes in volumes after mixing.
Therefore, check again how solvents are being mixed on a daily basis.
Procedure 1 - Preparing 1 L (approx.) of Mobile Phase
1) Measure 500 mL of ethanol using a graduated cylinder.
2) Measure 500 mL of water using a separate graduated cylinder.
3) Place both liquids in a bottle and shake thoroughly.
Procedure 2 - Preparing 1 L of Mobile Phase
1) Place 500 mL of ethanol in a 1 L volumetric flask.
2) Add water while stirring the flask.
3) Wait until the temperature returns to room temperature (temperature increases due to exothermic reaction).
4) Fill with water until the total volume is 1 L.
| Name | Definition |
| Molar Fraction | (number of moles of component of interest) ÷ (total number of moles of mixture) |
| Weight Fraction | (number of grams of component of interest) ÷ (total number of grams of mixture) |
| Volume Fraction | (volume of component of interest before mixing) ÷ (total volume of mixture) |