Chemistry Reference
In-Depth Information
Column lot or batch reproducibility should also be evaluated late in method devel-
opment or prevalidation; generally, method development is performed on one col-
umn lot, and then verified both on another new column from the same lot and a new
column from a different lot. These column lot evaluations are often performed either
as a part of intermediate precision or robustness (Chapter 4). Column lot reproduc-
ibility is less of an issue now compared to earlier, as many column manufacturers
now manufacture their columns from scratch instead of buying the base silica, which
itself can differ from lot to lot in trace impurities, which can affect chromatography.
One final note on the column front: it is also important that method development
be performed using only new HPLC columns as columns can have a memory effect
from previous conditions/methods that have been used, resulting in reproducibility
issues when the column is eventually replaced.
3.4.9 m
obIle
P
hASe
c
onSIderAtIonS
A rule of thumb for mobile phases used in method validation: the simpler the mobile
phase, the more robust it will be. Some of the common organic solvents and their
properties used in method development are listed in Table 3.4. Acetonitrile is gen-
erally preferred over methanol for method development because it has a lower UV
cutoff, resulting in better PDA spectral interpretations. Methanol may be preferred
for some MS applications; however, the actual selectivity obtained is more of a driv-
ing force for solvent choice. Use of high-temperature (reduced viscosity) and high-
pressure technology such as UHPLC (or both) has opened up the range of possible
tAble 3.4
properties of common mobile phase buffers and Additives
buffer or Additive
pK
a
*
buffer range
ms compatibility
Acetic acid (glacial)
4.8
Ammonium acetate p
K
a 1
4.76
3.8-5.8
Ye s
Ammonium acetate p
K
a 2
9.2
8.2-10.2
Ye s
Ammonium bicarbonate
9.2, 10.3
8.2-11.3
Ye s
Ammonium formate p
K
a 1
9.2
2.8-4.8
Ye s
Ammonium formate p
K
a 2
9.2
8.2-10.2
Ye s
Ammonium hydroxide
9.2
Ye s
Ammonium phosphate, dibasic
7.2, 9.2
6.2-10.2
No
Formic acid
3.8
Ye s
Phosphoric acid
2.1
No
Potassium phosphate, monobasic
2.1
1.1-3.1
No
Potassium phosphate, dibasic
7.2
6.2-8.2
No
Potassium phosphate, tribasic
12.7
11.7-13.7
No
Sodium citrate, tribasic
3.1, 4.8, 6.4
2.1-7.4
No
Triethylamine
11.0
Ye s
Triethylammonium acetate (TEEA) p
K
a 1
4.76
3.8-5.8
Ye s
Triethylammonium acetate (TEEA) p
K
a 1
11.0
10-12
Ye s
Trifluoroacetic acid
0.3
Ye s
