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Column Length

Just as reducing the slope of the gradient can increase chromatographic efficiency (N), thereby improving resolution and sensitivity, increasing the length of the column affords the same benefits.

The example of digested bovine serum albumin (BSA) demonstrates the huge resolving power achievable by coupling 8 x 250 mm columns in series to generate a 2 m long column (Figure 1).

Peptide map of HSA using a 2 m column

Figure 1: Peptide map of BSA using a 2 m column. 8 x 250 mm x 2.1 mm, 5 µm C18, 0.2 mL/min, gradient 0%A-60%B in 440 min, mobile phase A: 0.1% v/v TFA in 2% acetonitrile, mobile phase B: 0.1% v/v/ TFA in 70% acetonitrile, UV 214 nm, 60 °C (Sandra P. and Vanhoenacker G.J. Sep. Sci. 30 (2007) 241).


This is exemplified in greater detail when the HSA digest has been depleted of the six most abundant proteins (Figure 2). The exploded portion perfectly illustrates the fine detail achievable in what looks like a very heavily congested portion of the gradient.

Peptide map of HSA, after depletion of the six most abundant proteins , using a 2 m column

Figure 2: Peptide map of BSA, after depletion of the six most abundant proteins, using a 2 m column. 8 x 250 mm x 2.1 mm, 5 µm C18, 0.2 mL/min, gradient 0-70%B in 520 min, mobile phase A: 0.1% v/v TFA in 2% acetonitrile, mobile phase B: 0.1% v/v/ TFA in 70% acetonitrile, UV 214 nm, 60 °C (Sandra P. and Vanhoenacker G.J. Sep. Sci. 30 (2007) 241).


Peptide map analysis generated using 4 m, 2 m, 1 m and 0.25 m (250 mm) columns are shown in Figure 3, with their corresponding efficiency (N) vs. gradient time plots (Figure 4). The gains in efficiency as column length increases are clear, and it is also worth noting that the reduction in gradient slope has a similar benefit across column length. In addition, the benefits, in terms of increased efficiency with column length, dwindle as column length increases i.e. the gaps between the plots becomes closer.

Peptide maps with differing column lengths

Figure 3: Peptide maps with different column lengths.


Efficiency vs gradient time plots

Figure 4: Efficiency vs. gradient time plots for different column lengths.

 
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