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POSTED ON JANUARY 3, 2018 BY DR. DEEPAK BHANOT 1 COMMENT

Guidelines on use of bu ers in

HPLC separations
Bu ers play an important
role  in Reverse phase HPLC
separations. Changes in pH
in uence the degree of
ionization of sample molecules.
The change is minimal in case
of non-polar molecules but
assumes signi cance in case of
polar molecules which are
acidic or basic in nature.
Changing the pH can be used
e ectively to separate closely
spaced eluting peaks or may Scientist mixing solvents for preparation of bu er solution

even lead to merger of

separate peaks. It is for this
reason that a control of pH through use of bu ers helps improve separations of closely spaced
or overlapping peaks.
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There are several key factors that contribute to control of pH through use of bu ers which are TODAY WEEK MONTH ALL
brie y covered in the article.
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Bu er selection
The choice of the appropriate bu er for an application in hand is governed by bu er
characteristics such as pka,pH range and UV cut-o . The values for these parameters can be TOPICS
easily found in reference texts. As a general rule the pH bu ers should be used within +/- 1 unit
of its pka value. Within this range the bu ers resist any deliberate attempts to change pH. As an AAS (53)

example a bu er having pka value of 4.8 can be e ectively used over a pH range of 3.8-5.8.The Analytical Chemistry (3)
UV cut-o value should also be considered as the detection wavelength should not interfere with
Calculations (5)
the bu er absorbance.
Chemical (2)

Bu er pH Chromatography (9)

Silica based columns should not be used outside the pH range2.0-8-0.Below pH 2.0 there can be Environmental Analysis (2)
loss of the organic bonded phase and on the other hand pH values above 8.00 can result in
Featured (2)
greater solubility of silica backbone of stationary phase . For such applications columns with
other packings can prove to be useful. Free e-courses (2)

Gas Chromatography (55)

Bu er Concentration
General Topics (140)
Deciding on bu er concentration is important for method development. Ideally the lowest
Guest Posts (27)
concentration that gives reproducible results should be chosen. Higher concentration leads to
faster elution of polar molecules. Generally the bu er concentration should not be lower than HPLC (60)
0.005M. Below this concentration it may not remain as an active bu er. On raising bu er
ICP Techniques (13)
concentration there can be increase in bu er viscosity which can increase column back
pressures. Normally the concentration should be kept in the 0.005 to 0.1M range. Jobs (1)

Lab Bytes (17)

Bu er solubility Lab Management (95)

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28/04/2019 Guidelines on use of buffers in HPLC separations - Lab-Training.com

The bu er should be fully soluble in the dissolving media. At higher concentrations precipitation Laboratory Insurance (8)
can take place on coming in contact with the organic component of the mobile phase. Such
Laboratory Safety (13)
precipitation can lead to operational problems in pumps as well as blocking of the columns.
Method Validation (1)

Preparation of bu er solutions Microbiology (14)

Weigh the required quantity in the volumetric ask and start adding the required solvent. Adjust News (3)
the pH of the solution before lling the ask to the required mark. Adjustment of pH should be Paper Chromatography (10)
made before starting addition of the required organic phase. Next lter the solution through
0.45μm lter for routine analytical applications and 0.22μm lter for UHPLC applications. This is pH Measurement (3)

essential for removal of any residual solid suspensions. Bu ers after their preparation should be Preformulation Studies (3)
used at the earliest and if any solid or bacterial suspensions are observed before use they should
Quality System (9)
be discarded and prepared freshly.
Separation Science (11)
It can be seen that bu ers play a crucial role in majority of HPLC separations. Method
Spectroscopy (80)
development often requires careful selection of bu ers and adequate care in their preparation.
Stability studies (8)

Filed Under: HPLC Training (21)

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

Jarvis says:
February 7, 2018 at 6:21 pm

Refreshing.

Reply

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