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A Stable Method
Using 100% Aqueous Mobile Phase
Cleaning Validation of Guanidine
Figure 3: Cleaning validation example for residual guanidine. Upper chromatogram shows a standard at about 100 ppm, lower
shows a real life sample with a residue of about 10 ppm.
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printable Application Sheet
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Note: It may be necessary to remove guanidine from protein preparations, especially if they are to be used for therapeutic
appilcations Thus it is necessary to monitor low concentrations of guanidine. The Bidentate C18 offers a stable, reliable
method that produces low LOQ compared with ordinary silica based columns.
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Method Conditions
| Column |
Cogent Bidentate™, 4µm, 100A |
| Catalog No. |
40018-15P |
| Dimensions |
4.6 x 150 mm |
| Mobile Phase |
100% DI water + 0.5% phosphoric acid + 1.5g/L pentane sulfonic acid |
| Flow rate |
1.0 mL/minute |
| Peaks |
1: Impurity
2: Guanidine
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| Injection Volume |
20 µL |
| Detection |
UV 200 nm |
| Temperature |
25°C |
| Std Concentration |
100ppm |
| Sensitivity |
~1.6ppm as LOQ |
Discussion
Guanidine is a strong base, therefore it will be protonated at all pH values below 12 thus will carry a positive charge. The low
molecular weight of guanidine, with its positive charge, and lack of a significant chromophore, make the analysis very difficult.
Since guanidine is such a polar compound, it requires a 100% aqueous mobile phase to be retained in reverse phase on a C18 column.
Normally this mobile phase would require a specialty column such as a polar-embedded phase, but these columns are hydrolytically
unstable and have other issues while running the method. Since the Cogent Bidentate C18 does not suffer from loss of retention from
run to run which is commonly known as “phase collapse” and it is not a specialty column, it can be used as an L1 or reversed phase
C18 column. One of the advantages of this column is that it is hydrolytically stable under aggressive, acidic mobile phase conditions
as shown in this application note. Also, since it is still an L1 column, it could be interchanged with a C18 method column without
revalidation of the method. This separation works best with a highly acidified mobile phase.
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