The Cardinal Rules of 2DLC Method Development

Column selection Column selection must lead to a minimum correlation between retention mechanisms (orthogonality must be maximized). 

Sampling The second column method must be as fast as possible to allow for optimal sampling of the first dimension. The method development of the second dimension should be done first. The sampling rate for the second-dimension column should maintain three to four samples across the narrowest peak in the first dimension for optimum 2DLC resolution. Less than three samples across the narrowest peak in the first dimension allow for faster analyses with lower 2DLC resolution. 

Second-dimension elution time range The second-dimension elution time range must be determined. The flow rate needs to be optimized for maximum resolution and speed. This will establish the performance of the second dimension. The elution time range can be tuned with either gradient elution and/or by flow rate to determine the sampling rate. 

S ample loop volumes The volumes of the sample loops that store eluent from the first dimension and inject eluent into the second-dimension column system must be determined. The loop volume divided by the second-dimension elution time range determines the first-dimension flow rate in comprehensive 2DLC. If the dilution factor is small in the second column, a flow splitter can maintain a small loop volume even with a substantial flow rate from the first-dimension column. 

First-dimension optimization The flow rate, elution time range, and the efficiency of the first-dimension column must be carefully controlled and matched to the second-dimension column, the sample loop volume, and the sampling rate. 

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