Wednesday 8 June 2011

Interpreting a HPLC analytical method

A resource for chromatographers

The detail which is included in a HPLC analytical method will dictate how much interpretation is required by the analyst when following the method. For a very detailed method the analyst should be able to follow it easily but if the method does not include certain details the analyst will have to make some decisions about how to perform the analysis.

Local laboratory proceures
Routinely parts of the HPLC analytical method are covered by the local procedures in the analytical laboratory and thus will not be included in the HPLC analytical method. Examples are:
The sequence of the injections to be performed.
The procedure to use when preparing the mobile phase, e.g. the grades of solvents which should be used.
The routine use of guard columns or cartridges.
The implementation of wash programmes for column post analysis.

A good knowledge of the local laboratory procedures is required to ensure that all requirements are met.

HPLC column not defined or unavailable
When following a pharmacopoeia method, the column to use will be defined by the bonded phase type (e.g. octadecyl silane) and the particle size range (e.g. 1.5 to 10µm). This makes it difficult to choose which column to use since these parameters may be used to describe hundreds of columns. It is up to the analyst to select a suitable column. There may be a preference in the laboratory for a particular column to use in these situations. Some method development may be necessary to achieve the correct results following the monograph.

Another problem related to columns which may be encountered is that the column needed for a method is no longer available. In this case an equivalent column needs to be sourced. To find an equivalent column a column classification system is required. There are a number of researchers working in this area and their work has produced data for comparing columns [1,2].

Dwell volume
When using gradient methods the effects of dwell volume can lead to changes in the retention times when methods are transferred between different instruments. Ideally methods are developed to be robust to changes in dwell volume but if the change results in reduced resolution a solution will be required.If the retention times are shorter than expected due to the difference in dwell volume then the current system has a smaller dwell volume than the previous system. A solution is to introduce a ‘hold’ of the initial gradient mobile phase composition at the beginning of the analysis. The length of the hold can be determined by measuring the dwell volume on the two systems and dividing the difference by the flow rate, or, it can be determined experimentally by injecting some test samples with different hold values programmed into the gradient table.If the retention times are longer than expected due to the difference in dwell volume then the current system has a larger dwell volume than the previous system. Unless the injector has a function whereby the injection can be performed after the gradient has started (unlikely in older systems which are more likely to have a large dwell volume) the method cannot be satisfactorily run on the instrument.

The dwell volume of an HPLC system is easy to measure:
  1. Remove the column from the system and use a short length of 0.010″ tubing to connect the injector directly to the detector.
  2. For solvent A, use HPLC grade water; for solvent B, add about 0.1% acetone to water (methanol or acetonitrile can be used instead of water).
  3. Set the detector wavelength to 265nm.
  4. Run a typical gradient from 0 to 100% B (e.g. 0-100% in 10 minutes at 1 mL/min flow).
  5. Record the detector signal during this gradient.
  6. Print out or display the "chromatogram" from the gradient run. It should look like Figure 1. Draw the best straight line fit to the flat portion at the beginning of the plot. Draw the best straight line fit to the linear ramp of the gradient. The time at which these two lines intersect is the dwell time (tD). The dwell volume is the product of the dwell time and the flow rate used for the test.
Figure 1

1. D. Visky, E. Haghedooren, P. Dehouck, Zs. Kovács, K. Kóczián, B. Noszál, J. Hoogmartens, E. Adams, , J. Chromatogr. A, 1101, 103-114, 2006, ‘Facilitated column selection in pharmaceutical analyses using a simple column classification system’.(This research group provide their column classification data on this website:
2. M.R. Euerby, P. Petersson, J. Chromatogr. A, 994, 13-36, 2003, ‘Chromatographic classification and comparison of commercially available reversed-phase liquid chromatographic columns using principal component analysis’.This research group has a collaboration with Advanced Chemistry Development (ACD/Labs) to provide a column selection tool (

This blog post is an excerpt from 'An Introduction to HPLC for Pharmaceutical Analysis' by Oona McPolin, available to purchase through the MTS website.

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