Method Transfer between HPLC and UPLC using ACQUITY UPLC H-Class and Method Transfer Kits


The Application Notebook

The Application NotebookThe Application Notebook-07-02-2010
Volume 0
Issue 0
Pages: 43

Transferring LC methods between instruments and/or laboratory sites is a common practice that allows analytical scientists to maximize equipment usage.

Kenneth J. Fountain, Hillary Hewitson and Damian Morrison, Waters Corporation, Milford, Massachusetts, USA.


Transferring LC methods between instruments and/or laboratory sites is a common practice that allows analytical scientists to maximize equipment usage. Although common, method transfer can be a difficult task. Differences in the properties of the LC system can produce gradient profiles that are inconsistent between the original method and the transferred method. The selectivity of the column must also remain constant when transferring from one LC to another. Complexity is added when methods are transferred between different technology platforms such as HPLC and UPLC. In this application, a method for clozapine and four impurities is transferred from UPLC to HPLC using the ACQUITY UPLC H-Class system. Selectivity of the separation is preserved by using columns with the same stationary phase that differ only in particle size.


UPLC system: Waters ACQUITY UPLC H-Class system with PDA detector

HPLC system: Waters Alliance 2695 separations module with 2998 PDA detector

Columns: BEH Shield RP18 1.7 to 3.5 µm method transfer kit

Mobile phase A: H2O

Mobile phase B: Acetonitrile

Mobile phase C: 200 mM ammonium formate, pH 10

Sample: USP resolution mixture for clozapine (0.144 mg/mL in 80/20 MeOH/H2O)

Detection: 257 nm

Column temp.: 30 °C

Gradient, flow rate and injection volumes are specified in the figure captions. Calculations to preserve the gradient profile were performed with the new ACQUITY UPLC columns calculator.

Figure 1: Separation of clozapine and its impurities on (a) the ACQUITY UPLC H-Class system and (b) the Alliance 2695 HPLC system. UPLC gradient is from 5 to 95% B in 5 min with a constant flow of 5% C. HPLC gradient holds at 5% B for 1.91 min and then goes to 95% B in 20.6 min with a constant flow of 5% C. UPLC flow rate is 0.5 mL/min and injection volume is 2.5 µL; HPLC flow rate is 1.17 mL/min and injection volume is 24 µL.

Results and Discussion

Figure 1(a) shows the separation of the clozapine sample on a 2.1 × 50 mm, 1.7 µm BEH Shield RP18 column at high pH. All impurities were sufficiently resolved from the main active ingredient. This method was then transferred to an HPLC system using a column with identical resolving power and selectivity [Figure 1(b)]. The system dwell volumes were measured and input into the ACQUITY UPLC columns calculator to maintain the same gradient profile. Note that the selectivity between the two separations is identical and the relative retention times of each impurity to clozapine is maintained (Table 1).

Table 1: Relative retention times of impurities to clozapine for HPLC and UPLC separations.


The combination of the ACQUITY UPLC H-Class system and column method transfer kits facilitates the transfer of LC methods between HPLC and UPLC platforms. This allows methods to be easily transferred between systems, laboratories and sites across the world without compromising separation performance.

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