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Solvents Mixing Ratio in Liquid Chromatography

Part 1. Mobile Phase: How Would You Make This Solution?

Usually, an HPLC solvent is mixed in terms of relative volumes (v/v) or relative weights (w/w). We have to remember that the volume of a solution will vary at different temperatures, therefore mixing solvents using relative weights ensures a high level of reproducibility of the mixture preparation. Due to the fact that this procedure is simply more awkward the mixing of solvents using relative volume seems to be more common. Consequently, if the mixing method is not specified in test method / application, it is reasonable to assume that mixing is performed by relative volume. In some cases, like viscous solutions such as amine, it would be desirable to mix using weight relative to volume (w/v) rather than the relative volumes, unless you will account for the specific gravity of the solutions at a given temperature to accurately measure the volumes.
Literature and HPLC data (white papers, pharmacopoeias, etc.) display a wide variety of conventions for mobile phase conditions used in HPLC data acquisition. In very rare cases, precise instructions of the method used to prepare the mobile phase are stated, such as “Add 340 L of phosphoric acid to 100 mL of water…”. Sometimes descriptions such as “20%-acetonitrile aqueous solution” or “acetonitrile/water (40/60)” are given, and sometimes descriptions containing proportions that do not add up to 100%, such as “acetonitrile/water (21/5)” or “methanol / water / phosphoric acid (95/5/0.3)” are given.
In any case, although there is no definitive international convention for the composition of mobile phases, it is necessary to use notation which ensures mobile phases are prepared under the same conditions when performing analysis every time. What is even more important is the understanding of this notation.

Part 2. Meaning and Preparation of “50% (v/v) Ethanol Aqueous Solution”

The description of Mobile Phase such as “ethanol/water (1/1)” would probably be handled using the preparation method described in “Procedure 1” below. Should the Mobile Phase description be “50%-ethanol aqueous solution”, most people would still probably use Procedure 1.

Procedure 1 - Solvents Mixing Ratio in HPLC

A dictionary of chemical terms indicates that this is a case of “volume percentage (% v/v)”, for which Procedure 2 is the more accurate for the mobile phase preparation. This means that the mobile phase compositions resulting from a percentage indication and a relative volume indication (e.g., “1:1”) would be different if not accounted for the mixtures properties.

Procedure 2 - Solvents Mixing Ratio in HPLC

The density of an HPLC solvent mixture is not the same as the simple average of the densities of the original mixture components, therefore the compositions of mobile phases prepared with the above methods can be different. For example, if 50 mL of water and 50 mL of ethanol were mixed together at around room temperature (25 °C), the resulting volume would only be 96 mL, not 100 mL as suspected.

In general, Procedure 1 is widely used as it is relatively simple. Due to this fact it is recommended that the “AAA/BBB (2/3)” style of notation is most commonly used.

Part 3. Solvent volume and temperature

The density of a solution/liquid is impacted by the surrounding ambient temperature. The temperature of a solution that has just been taken out of storage is sometimes significantly lower than the ambient room temperature in the laboratory, and mixtures of alcohols (i.e. methanol, ethanol) and water become warmer due to the exothermic reaction. For this reason, in order to prepare mobile phases with a high degree of reproducibility and accuracy, it is recommended that, before use, solutions/solvents once taken from storage units are left to reach the room temperature. Unless, of course, the storage unit if directly within the laboratory and the storage temperatures are no different to the ambient room temperature. Another very important fact is that once you achieve the required chromatography you should always follow the same mobile phase preparation procedure to the letter.

Part 4. Case Study – Methanol/Water mix – is 40% v/v the same as 40% w/w?

TaskCalculate equivalent concentrations from %v/v to %w/w and compare the result
Solution 1

(% v/v)

Preparation:400mL of methanol was mixed with 600mL of water at 25oC
Conc. (%v/v)40% methanol   &   60% water  (v/v)
Calculation of Conc. (%w/w)Methanol density at 25oC:       0.791 g/mL

Water density at 25oC:              0.997 g/mL



400mL of Methanol = 400 mL x 0.791 g/mL     = 316.4 g

600mL of Water        = 600 mL x 0.997 g/mL     = 598.2 g

Total weight of solution = 914.6 g


% w/w calculation:

–          Methanol % w/w    = 316.4 g / 914.6 g x 100     = 34.6 % w/w

–          Water % w/w            = 598.2 g / 914.6 g x 100     = 65.4 % w/w



40/60 (% v/v) solution of methanol/water contains 34.6 % w/w of Methanol and 65.4% w/w of Water !!!

Solution 2

(% w/w)

Preparation:400 g of methanol was mixed with 600g of water at 25oC
Conc. (%v/v)40% methanol   &    60% water   (w/w)
ReferencesMethanol for HPLC information – https://www.sigmaaldrich.com/IE/en/product/sigald/34860

Pure Water information – https://www.sigmaaldrich.com/IE/en/product/sial/denwat

There is only one conclusion from the above 40/60 %v/v is NOT equivalent to 40/60 %w/w.

Part 5. Mixing Solvents with Two Pumps

Isocratic methods with organic solvent and water are most commonly used in reversed-phase analysis. With such methods, the retention times when the two types of mobile phase are delivered with (1) two pumps using a high pressure 2-liquid gradient system and then mixed in a closed system using a HPLC built in mixer, and (2) when the two types of mobile phase are mixed by analyst in a bottle and delivered with one pump differ due to the change in volume after mixing. You must remember that technology is there to help you overcome some physio-chemical properties of solvents and use compressibility settings when using 2x separate pumps to mix the solvents on the system.

Take this opportunity to check what kind of method is used to mix an HPLC solvent in your daily analysis work and please do not hesitate to contact us should you have any questions regarding the above.

Source: Tips for practical HPLC analysis – Separation Know-how. LC World Talk Special Issue Volume 2. Shimadzu.

Liquid Chromatography – Master the Basics

This article is part of our “Liquid Chromatography – Master the Basics” series, your go-to resource for comprehensive and insightful updates on the world of liquid chromatography. Each month in 2024 we will dive into a Liquid Chromatography topic, offering content that is both accessible to beginners and beneficial for experienced scientists.

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Sebastian Jurek is an application consultant with Mason Technology with specialist knowledge in the Shimadzu range of instrumentation. He holds more than 22 years experience in chromatography techniques and analytical method development, optimisation and troubleshooting.

Get in touch with Sebastian today if you would like further information on our range of Shimadzu products.

Sebastian-Jurek-application-consultant-with-Mason-TechnologySebastian Jurek
Application Consultant for Shimadzu Chromatography
E:      sjurek@masontec.ie
M:     +353 87 436 4185
T:   +353 1 4154422

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