APPENDIX II

PREPARING BUFFERED
MOBILE PHASES

II.1

SEQUENCE OF OPERATIONS

Buffered mobile phases can be prepared by the following sequence of operations:

1. combine the buffer ingredients with water to obtain the aqueous buffer
(solution A)
2. confirm or adjust the pH of solution A with a pH meter
3. combine a given volume (e.g., 200 mL) of organic buffer (solution B) with
a given volume (e.g., 800 mL) of solution A from step 2 to obtain the final
mobile phase (20% organic buffer in this example)
4. check the pH of the final mobile phase (optional)
Because a pH measurement for a mobile phase that contains organic buffer is
unreliable due to drift of the pH meter, step 4 is only useful for detecting major
errors in the formulation or comparing two solutions with the same organic content.
Most laboratories elect to skip step 4.
The usual approach in step 1 is to formulate aqueous buffers of differing pH
(A1 and A2), and then combine these two solutions in the correct proportions to
obtain final solution A with the desired pH. If the pH is adjusted in step 2, the
same two starting solutions can be used to titrate the final buffer to the desired
pH as measured by the pH meter. The precision of a pH measurement (step 2)
in most laboratories is usually no better than 0.05 to 0.10 pH unit, which can
cause significant changes in resolution for some samples (Section 7.3.4.1). When
an HPLC method is pH sensitive, step 2 should be used only for an approximate
confirmation of pH. By combining accurate weights of the buffer ingredients with
accurate volumes of distilled and degassed water (without further adjusting pH),
the pH of the buffer solution can be controlled within narrow limits (0.02 unit).
Buffer concentrations whose pH is known quite accurately are also commercially
available.
Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder,
Joseph J. Kirkland, and John W. Dolan
Copyright 2010 John Wiley & Sons, Inc.

887

888

PREPARING BUFFERED MOBILE PHASES

Table II.1
Preparation of Low-pH Phosphate Buffers of Defined pH
Required pH

Volume (mL) of A1a

Volume (mL) of Ab

2.0

565

435

2.2

455

545

2.4

345

655

2.6

250

750

2.8

175

825

3.0

110

890

3.2

55

945

a Solution

of 0.1 M phosphoric acid; the phosphoric acid used to prepare this stock solution must be
titrated to confirm the amount of phosphoric acid present.
b Solution of 0.1 M sodium monophosphate; combine 13.8 g of NaH PO monohydrate with water in a 1-L
2
4
flask.

It is common practice to adjust the buffer pH with a concentrated acid. For

example, solution A2 of Table II.1 might be prepared and titrated to the desired pH
with concentrated phosphoric acid. This still produces a buffer at the desired pH, but
the ionic strength of the buffer will be higher than if equimolar solutions of A1 and
A2 are blended. While it is unlikely to make much difference in the chromatographic
results obtained by the two techniques (titrating with concentrated acid vs. equimolar
blending) for RPC, some separations can be sensitive to differences in ionic strength
(especially ion exchange). It is best to describe in the method documentation exactly
how a buffer is to be prepared, and to follow these directionsconsistency in
mobile-phase preparation is generally important and will give more reliable results.
Acidic or basic additives are sometimes added to the mobile phase for various
purposes. When such additives are not used as the primary buffer, they should be
added to the desired quantity (concentration) of the buffer first; the mixture should
then be adjusted to the desired pH by titrating with acid or base.

II.2

RECIPES FOR SOME COMMONLY USED BUFFERS

The pH of a buffered solution remains approximately constant as the buffer is

diluted or concentrated, or when one ionized cation (e.g., Na+ , K+ ) or anion (e.g.,
Cl , Br ) is replaced by another. Tables II.1 to II.3 describe the preparation of
some buffers that are commonly used in RPC (adapted from [1])using the mixing
of two solutions A1 and A2, each of which have an equal concentration of the
buffering species. The specified volumes of solutions A1 and A2 are combined and
mixed to give the final buffer solution of a required pH. The formulations of Tables
II.1 to II.3 are based on a final buffer concentration of 0.1M and sodium as cation.
Formulations for other buffer concentrations and/or the use of different cations (K+
is usually preferred) can be inferred from these data. The pH of buffers that are more
dilute or more concentrated, or that contain different cations, may differ slightly

II.2 R E C I P E S F O R S O M E C O M M O N L Y U S E D B U F F E R S

889

Table II.2
Preparation of Acetate Buffers of Defined pH
Required pH

Volume (mL) of A1a

Volume (mL) of A2b

3.6

926

74

3.8

880

120

4.0

820

180

4.2

736

264

4.4

610

390

4.6

510

490

4.8

400

600

5.0

296

704

5.2

210

790

5.4

176

824

5.6

96

904

Solution of 0.1 M acetic acid; combine 6.0 g (5.8 mL) of glacial acetic acid with water in a 1-L flask.
of 0.1 M sodium acetate; combine 8.2 g of sodium acetate (or 13.6 g sodium acetate trihydrate)
with water in a 1-L flask.

b Solution

Table II.3
Preparation of Intermediate-pH Phosphate Buffers of Defined pH
Required pH

Volume (mL) of A1a

Volume (mL) of A2b

5.6

948

5.8

920

80

6.0

877

123

6.2

815

185

6.4

735

265

6.6

685

315

6.8

510

490

7.0

390

610

7.2

280

720

7.4

190

810

7.6

130

870

7.8

85

915

8.0

53

947

a solution

52

of 0.1 M monobasic sodium monophosphate; combine 13.8 g of monobasic sodium monophosphate monohydrate with water in a 1-L flask.
b solution of 0.1 M dibasic sodium phosphate; combine 26.8 g of Na HPO .7H O with water in a 1-L flask.
2
4
2

890

PREPARING BUFFERED MOBILE PHASES

from these values. The exact pH value of the mobile phase is usually unimportant
in method development. What is important is that the final pH of the mobile phase
can be reproduced (preferably within 0.02 unit) each time a new batch of mobile
phase is prepared. Note that solutions only buffer effectively 1 pH unit from the
pKa value of the ionizable constituent (Section 7.2.1). Although the mobile phase
may be used at a temperature other than ambient, the pH at ambient is assumed
for the buffers of Tables II.1 to II.3 and should be used to describe the final mobile
phase.
As an alternative to Tables II.1 to II.3 as guides for buffer preparation, many
on-line buffer calculators are available (search for HPLC buffer calculator) that
provide for the use of several additional buffers. For example, one such calculator
(The Buffer Wizard, Zirchrom, Anoka, MN, www.zirchrom.com) provides buffer
preparation instructions. Input the acid, base, desired buffer concentration, and pH,
and the calculator provides instructions for preparation, along with warnings about
buffer capacity, column stability, and so forth.

REFERENCE
1. G. Gomori, in Meth. Enzymology, S. P. Colowicxk and N. O. Kaplan, eds., Academic
Press, New York, 1955, p. 145.