APPENDIX IV

PREPARING BUFFERED MOBILE PHASES

Buffered mobile phases can be prepared by the following sequence of opera-tions:

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 unrelia-ble 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 confir-mation of pH. By combining accurate weights of the buffer ingredients with accurate volumes of distilled and degassed water (without further adjusting

735

Practical HPLC Method Development, Second Edition by Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajch

Copyright 1997 John Wiley & Sons, Inc.

736 APPENDIX IV

TABLE IV.l Formulation of Low-pH Phosphate Buffets (25C) of Some Desired pH

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 NaH2P04 - H 2 0 with water in a 1-L flask, dissolve, dilute to volume and mix thoroughly.

pH Desired Volume (mL) of Al Volume (mL) of A2

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

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 C2H302Na (or 13.6 g of C2H302Na 3H20) with water in a 1-L flask, dilute to volume, and mix thoroughly.

pH Desired Volume (mL) of Al Volume (mL) of A2

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 2 % 704 5.2 210 790 5.4 176 824 5.6 % 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 2H20 with water in a 1-L flask, dilute to volume, and mix thoroughly.

pH Desired

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

Volume (mL) of Al

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 H20 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 7H20 (or 35.9 g of Na2HP04 - 12H20) with water in a 1-L flask, dilute to volume, and mix thoroughly.

pH Desired

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

Volume (mL) of Al

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

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

Volume (mL) of A l

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

Volume (mL) of A2

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 differ-ent 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

APPENDIX IV 739

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

8.6 8.8 9.0 9.2 9.4 9.6 9.8

10.0 10.4 10.6

Volume (mL) of Al

25 25 25 25 25 25 25 25 25 25

Volume (mL) of A2

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 (prefera-bly 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).