Practical HPLC Method Development, Second Edition

by Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajch

Copyright 1997 John Wiley & Sons, Inc.

APPENDIX IV
PREPARING BUFFERED
MOBILE PHASES

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 (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 is unreliable due to drift of the pH meter, step 4 above is only useful for detecting
major errors in the formulation. Most laboratories elect to skip step 4.
The usual approach in step 1 is to formulate aqueous buffers of differing
pH (AI and A2), then combine these two solutions in the right proportions
to obtain solution A with the desired pH. If the pH is adjusted in step 2, the
same two 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 unit, which can
cause significant changes in the resolution of some samples. 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
735

736

APPENDIX IV

TABLE IV.l
Desired pH

Formulation of Low-pH Phosphate Buffets (25C) of Some

Solution Al: 0.1 M phosphoric acid. For accurate buffer formulations, the phosphoric
acid used to prepare this buffer must be titrated for the amount of H3PO4 present.
Solution A2: 0.1 M monobasic sodium monophosphate. Combine 13.8 g of NaH 2 P0 4
- H 2 0 with water in a 1-L flask, dissolve, dilute to volume and mix thoroughly.
pH Desired

Volume (mL) of A l

Volume (mL) of A2

2.0
2.2
2.4
2.6
2.8
3.0
3.2

565
455
345
250
175
110
55

435
545
655
750
825
890
945

Source: G. Gomori, in Meth. Emymology 1, S. P. Colowick and N. O. Kaplan, eds.. Academic

Press, New York (1955) 145.
TABLE IV.2

Formulation of Acetate Buffers (25*C) of Some Desired pH

Solution Al: 0.1 M acetic acid. Combine 6.0 g (5.8 mL) of glacial acetic acid with
water in a 1-L flask, dilute to volume, and mix thoroughly.
Solution A2:0.1 M sodium acetate. Combine 8.2 g of C 2 H 3 0 2 Na (or 13.6 g of C 2 H 3 0 2 Na
3H 2 0) with water in a 1-L flask, dilute to volume, and mix thoroughly.
pH Desired

Volume (mL) of A l

Volume (mL) of A2

3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6

926
880
820
736
610
510
400
2%
210
176
%

74
120
180
264
390
490
600
704
790
824
904

Source: G. Gomori, in Meth. Emymology I, S. P. Colowick and N. O. Kaplan, eds.. Academic

Press, New York (1955) 145.

pH), the pH of the buffer solution can be controlled within narrow limits
(0.02 unit). Buffer concentrates whose pH is known quite accurately are
also commercially available.
Acids or bases (e.g., triethylamine, acetic acid) are sometimes added to the
mobile phase as a means of improving peak shape and plate number (Section

TABLE IV.3

Formulation of Citrate Buffers (25*) or Some Desired pH

Solution Al: 0.1 M citric acid. Combine 21.0 g of citric acid with water in a 1-L flask, dilute to
volume, and mix thoroughly.
Solution A2: 0.1 M sodium citrate. Combine 29.4 g of QHsC^Naj 2H 2 0 with water in a 1-L
flask, dilute to volume, and mix thoroughly.
p H Desired

Volume (mL) of Al

3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0

930
870
810
750
700
660
610
560
510
460
410
360
320
270
230
190
140
60
40
30
15

Volume (ml

70
126
190
260
300
340
370
440
490
540
590
640
680
726
764
810
856
940
960
970
985

Source: G. Gomori, in Meth. Enzymology I, S. P. Colowick and N. O. Kaplan, eds.. Academic

Press, New York (1955) 145.

TABLE rV.4 Formulation of Intermediate-pH Phosphate Buffers (25C) of Some

Desired pH
Solution Al: 0.1 M monobasic sodium monophosphate. Combine 13.8 g of NaH2PO H 2 0 with
water in a 1-L flask, dilute to volume, and mix thoroughly.
Solution A2: 0.1 M dibasic sodium phosphate. Combine 26.8 g of Na2HP04 7H 2 0 (or 35.9 g
of Na2HP04 - 12H20) with water in a 1-L flask, dilute to volume, and mix thoroughly.
pH Desired

Volume (mL) of Al

5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0

948
920
877
815
735
685
510
390
280
190
130
85
53

Volume (ml

52
80
123
185
265
315
490
610
720
810
870
915
947

Source: G. Gomori, in Meth. Enzymology I, S. P. Colowick and N. O. Kaplan, eds., Academic

Press, New York (1955) 145.

738

APPENDIX IV

TABLE IV.5 Formulation of High-pH Tris" Buffers (25C) of Some Desired pH

Solution Al: 0.1 M Tris (free base). Combine 12.11 g of Tris with water in a 1-L flask,
dilute to volume, and mix thoroughly.
Solution A2: 0.1 M HCl. Obtain a 0.1M HC1 solution or prepare by appropriate
dilution of a stronger solution.
pH Desired

Volume (mL) of A l

Volume (mL) of A2

7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9

50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50

45.7
44.7
43.4
42.0
40.3
38.5
36.6
34.5
32.0
29.2
26.2
22.9
19.9
17.2
14.7
12.4
10.3
8.5
7.0

Source: R. G. Bates and V. E. Bower, Anal. Chem., 28 (1956) 1322.

Tris(hydroxymethyl)aminomethane
7.3.3.2). When these additives are not used as the primary buffering agent,
they should be added to the desired quantity (concentration) of the buffer
first; then the mixture should be adjusted to the desired pH by titrating with
acid or base.
IV.l

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 (Na + , K + ) or anion
(Cl", Br") is replaced by another. The formulations of Tables IV.l to IV.4
are based on a final buffer concentration of 0.1 M and sodium as cation;
however, formulations for other buffer concentrations and/or the use of different cations (potassium is usually preferred) can be inferred from these data.
The pH of buffers that are more dilute or more concentrated, or which contain
different cations may differ slightly from these values. The exact pH value of

739

APPENDIX IV

TABLE rv.6 Formulation of High-pH Glydne Buffers (25C) of Some Desired

PH
Solution Al: 0.2 M Glycine. Combine 15.01 g of Glycine with water in a 1-L flask,
dilute to volume, and mix thoroughly.
Solution A2:0.2 M NaOH. Obtain a 0.2 M NaOH solution or prepare by appropriate
dilution of a stronger solution.
pH Desired

Volume (mL) of Al

Volume (mL) of A2

8.6
8.8
9.0
9.2
9.4
9.6
9.8
10.0
10.4
10.6

25
25
25
25
25
25
25
25
25
25

2.0
3.0
4.4
6.0
8.4
11.2
13.6
16.0
19.3
22.75

Source: O. Gomori, in Meth. Enzymology I, S. P. Colowick and N. O. Kaplan, eds., Academic

Press, New York (1955) 145.
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 pi of the
ionizable constituent (e.g., acetate with a pi = 4.6 is an adequate buffer in
the range pH 3.6 to 5.6; see Table IV.2).
Tables IV.5 to IV.6 show formulations for two organic-based buffers which
are especially useful in the range pH 7 to 10.6. These organic buffers may be
particularly useful to minimize silica-based column degradation (see Sections
5.4.3.6, 7.2.2.4, and 11.2.3).