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POSTED ON MAY 9, 2014 BY DR. DEEPAK BHANOT 2 COMMENTS

How to Mix HPLC Mobile Phase

Solvents for consistency of
results?
A single solvent seldom meets
the requirements for
separations carried out using
HPLC. In most cases two or
more solvents are required to
be mixed. In isocratic
operation the composition is
pre- decided and remains
unchanged during the
analytical run. On the other
hand in gradient elution the
nal solvent composition is a
lot di erent from the initial Mixing of Solvents
composition and changes are
programmed during the
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In isocratic mode solvent mixtures are prepared and mixing is carried out o -line Considerations Jetpack plugin with Stats module
for mixing of HPLC mobile phase solvents o -line  discusses the role of physical properties of needs to be enabled.
individual solvents to be mixed. The present article discusses the importance of adopting same
mixing practice for ensuring consistency of results.

TOPICS
Stoichiometric mixing is an objective which is seldom achieved. Even when volumetric
measurement errors are eliminated heat of mixing prevents exact mixing of solvents. Energy in AAS (53)
the form of heat is either absorbed or liberated during the physical mixing of solvents. This
Analytical Chemistry (3)
results in temperature changes which lead to changes in volume of mixing of solvents. Methanol
and acetonitrile are two commonly used solvents in HPLC separations. Methanol on mixing with Calculations (5)
water results in libetration of heat which can increase the volume after mixing. On the other
Chemical (2)
hand acetonitrile on mixing with water results in absorption of heat resulting in cooling and
contraction of mixed volume. Such composition variations result in noticeable changes of polarity Chromatography (9)
of mixtures which have observable variations in separation characteristics of sample molecules.
Environmental Analysis (2)

Featured (2)
Mixing Practices for Solvents
Free e-courses (2)
Mixing of solvents is an elementary operation which is picked up easily. Two mixing practices
commonly adopted are: Gas Chromatography (55)

General Topics (140)

Separate volumetric measurements
Guest Posts (27)

As an example suppose you are required to prepare a one liter mixture of methanol and water HPLC (60)
having 60% water and 40% methanol. In this method 600 ml of water are measured in a
ICP Techniques (13)
graduated cylinder and transferred to a mixing beaker. Next 400 ml of methanol is measured in
the graduated cylinder and is transferred to the water in the mixing beaker and the mixture is Jobs (1)
stirred for even mixing. Due to heat of mixing temperature rise will result in nal volume above Lab Bytes (17)
1000ml.
Lab Management (95)

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Make-up of mixed volume Laboratory Insurance (8)

Laboratory Safety (13)

In this method you would measure 600 ml of water in a graduated cylinder and then make up
Method Validation (1)
volume to 1000 ml mark with methanol. The total volume is then transferred to the mixing
beaker for uniform mixing. Microbiology (14)

News (3)
Theoretically both the methods serve the requirements of mixing solvents but the volumetric
variations are found to be di erent for both the methods. It is therefore important to adopt the Paper Chromatography (10)
same mixing method for consistency of separations. In case of bu ers pH is adjusted in the
pH Measurement (3)
aqueous phase prior to mixing with the organic phase.
Preformulation Studies (3)

In case of online mixing pure mobile faces are kept in separate bottles and mixed in the desired Quality System (9)
proportion with the help of pomp proportionating valves. Near stoichiometric mixing is possible
Separation Science (11)
after corrections for heat of mixing are applied through the software.
Spectroscopy (80)
Hopefully the article has served to emphasize the need for adopting the same mixing procedure
Stability studies (8)
every time. Please show and share your opinion and o er your valued comments.
Training (21)

Filed Under: HPLC Tagged With: High-performance Liquid Chromatography, Hplc, Mobile Phase Weighing (3)

About Dr. Deepak Bhanot

Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise
beginning from sales and product support of analytical instruments. After
completing his graduation and post graduation from Delhi University and IIT
Delhi he went on to Loughborough University of Technology, UK for doctorate research in
analytical chemistry. His mission is to develop training programs on analytical techniques and
share his experiences with broad spectrum of users ranging from professionals engaged in
analytical development and research as well as young enthusiasts fresh from academics who
wish to embark upon a career in analytical industry.

Comments

A. Garcia says:
August 4, 2017 at 12:26 am

Hallo Dr. Bhanot. First of all thank you very much for your useful and interesting
posts on HPLC. I have a question regarding the topic of mixing solvents in isocratic
methods: what is better, mix them o ine or online? I learnt that it is more adecuate
o ine due to the e ects of temperature and volume changes that may badly
in uence the chromatography and damage the pumps in long term and so on.
However, another approach is that pumps mix more accurate than people and thus
isocratic HPLC will be performed by mixing solvents in the mixing chamber. Thank
you very much in advance!

Reply

Dr. Deepak Bhanot says:

August 4, 2017 at 10:01 am

lab-training.com/2014/05/09/how-to-mix-hplc-mobile-phase-solvents-for-consistency-of-results/ 2/3
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Many thanks Garcia for appreciating our e orts.Online mixing is preferable due to
its accuracy over o ine mixing. The software on modern systems compensates
for volume changes, if any. The temperature changes are not signi cant enough to
a ect the chromatography or damage any internal components.

Reply

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