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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
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Academic Press. the phosphoric acid used to prepare this buffer must be titrated for the amount of H3PO4 present.8 5. Gomori. Colowick and N. pH Desired Volume (mL) of A l Volume (mL) of A2 3. O. Combine 8. O. acetic acid) are sometimes added to the mobile phase as a means of improving peak shape and plate number (Section . P.6 2.4 4.2 Formulation of Acetate Buffers (25*C) of Some Desired pH Solution Al: 0. New York (1955) 145. pH Desired Volume (mL) of A l Volume (mL) of A2 2. For accurate buffer formulations.2 2. S. Buffer concentrates whose pH is known quite accurately are also commercially available. S. Academic Press.0 g (5. Emymology I. New York (1955) 145.6 3. Emymology 1.1 M acetic acid. in Meth. Solution A2:0.. and mix thoroughly.8 mL) of glacial acetic acid with water in a 1-L flask. TABLE IV.4 5.0 3.l Desired pH Formulation of Low-pH Phosphate Buffets (25°C) of Some Solution Al: 0.2 565 455 345 250 175 110 55 435 545 655 750 825 890 945 Source: G.1 M phosphoric acid. Colowick and N.8 3.736 APPENDIX IV TABLE IV. Kaplan.1 M monobasic sodium monophosphate.1 M sodium acetate..4 2. Gomori. dilute to volume.2 4.0 4. in Meth.8 g of NaH 2 P0 4 . dissolve. dilute to volume and mix thoroughly.g. triethylamine. and mix thoroughly. the pH of the buffer solution can be controlled within narrow limits (±0.0 2.8 4. eds. Combine 13. eds. Acids or bases (e. Kaplan.2 5.2 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.0 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.6 4. dilute to volume. pH). Combine 6. P. Solution A2: 0..H 2 0 with water in a 1-L flask.02 unit).

Academic Press.4 3.3 Formulation of Citrate Buffers (25*) or Some Desired pH Solution Al: 0.2 6.4 7. O. Solution A2: 0.0 6.2 3.1 M sodium citrate..12H20) with water in a 1-L flask. in Meth.8 6. Combine 21.2 4.6 5. Combine 13.2 6.4 5. Academic Press. and mix thoroughly. New York (1955) 145. Gomori. Combine 29. S.0 5. Gomori.4 Formulation of Intermediate-pH Phosphate Buffers (25°C) of Some Desired pH Solution Al: 0. O. dilute to volume. Kaplan.8 8.0 930 870 810 750 700 660 610 560 510 460 410 360 320 270 230 190 140 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.0 7.1 M citric acid.6 4.4 4. Colowick and N.4 6. in Meth.0 6.8 7. and mix thoroughly.4 6. pH Desired Volume (mL) of Al 5. Combine 26.1 M dibasic sodium phosphate. eds.0 g of citric acid with water in a 1-L flask. S.TABLE IV.0 4.8 7.6 6. Enzymology I. Enzymology I.6 3. and mix thoroughly.2 5. P.2 7.8 4. P.6 5.8 5.6 7. Colowick and N..8 g of NaH2PO« • H 2 0 with water in a 1-L flask.4 g of QHsC^Naj ■ 2H 2 0 with water in a 1-L flask. TABLE rV. Kaplan. and mix thoroughly. New York (1955) 145. . p H Desired Volume (mL) of Al 3.6 6.9 g of Na2HP04 .8 6. Solution A2: 0. dilute to volume. dilute to volume.0 3.0 948 920 877 815 735 685 510 390 280 190 130 85 53 Volume (ml 52 80 123 185 265 315 490 610 720 810 870 915 947 Source: G. dilute to volume. eds.8 g of Na2HP04 • 7H 2 0 (or 35.1 M monobasic sodium monophosphate.

The formulations of Tables IV.11 g of Tris with water in a 1-L flask. or which contain different cations may differ slightly from these values. dilute to volume. Chem. E.0 40. Bower.7 43. Anal. G.6 7.7 7.0 29.4 are based on a final buffer concentration of 0. and mix thoroughly.l RECIPES FOR SOME COMMONLY USED BUFFERS The pH of a buffered solution remains approximately constant as the buffer is diluted or concentrated. IV.2).7 12.1 M Tris (free base).0 8.2 7.1 7.738 APPENDIX IV TABLE IV. formulations for other buffer concentrations and/or the use of different cations (potassium is usually preferred) can be inferred from these data. then the mixture should be adjusted to the desired pH by titrating with acid or base. Obtain a 0.5 32.5 36. Solution A2: 0.7 44.1 M HCl.9 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 45.1M HC1 solution or prepare by appropriate dilution of a stronger solution.5 Formulation of High-pH Tris" Buffers (25°C) of Some Desired pH Solution Al: 0.6 34.3 7.3 8. K + ) or anion (Cl". Bates and V. °Tris(hydroxymethyl)aminomethane 7.8 8. The pH of buffers that are more dilute or more concentrated.0 Source: R.9 19. they should be added to the desired quantity (concentration) of the buffer first.2 14. When these additives are not used as the primary buffering agent.3 38.5 7.3.4 7.5 7. Combine 12.5 8. Br") is replaced by another.1 8.3.3 8.1 M and sodium as cation.7 8.4 8.6 8. pH Desired Volume (mL) of A l Volume (mL) of A2 7.4 42.l to IV. however. 28 (1956) 1322..8 7.2 22.2 8.9 17. The exact pH value of . or when one ionized cation (Na + .9 8.4 10.2 26.

2 M NaOH. Tables IV.6 16. dilute to volume. Academic Press. and 11. What is important is that the final pH of the mobile phase can be reproduced (preferably within ±0.6 8.5 to IV.2 13. Note that solutions only buffer effectively ±1 pH unit from the pi of the ionizable constituent (e.4.2 M NaOH solution or prepare by appropriate dilution of a stronger solution.01 g of Glycine with water in a 1-L flask.6 show formulations for two organic-based buffers which are especially useful in the range pH 7 to 10.2 9.6. Kaplan. eds.3. O.0 10. . Enzymology I.8 10. Colowick and N. 7.3 22.2).g.3).4 10.4.0 4. acetate with a pi = 4.4 11. New York (1955) 145.2.6 9.6.739 APPENDIX IV TABLE rv.6 Formulation of High-pH Glydne Buffers (25°C) of Some Desired PH Solution Al: 0.6.6 to 5. Obtain a 0.6 25 25 25 25 25 25 25 25 25 25 2.4 6.4 9.0 19.75 Source: O. the mobile phase is usually unimportant in method development. These organic buffers may be particularly useful to minimize silica-based column degradation (see Sections 5. S.0 9. Combine 15. P. in Meth.0 8.6 is an adequate buffer in the range pH 3..8 9.2 M Glycine. Solution A2:0.2.2.0 3.02 unit) each time a new batch of mobile phase is prepared. see Table IV. Gomori. and mix thoroughly. pH Desired Volume (mL) of Al Volume (mL) of A2 8..