Bio-Rad Chromatography Standards - Manual (Page 4)

1.2 Recommended Volume of Standard
The general recommendation is to load no more than 1% of the
column volume to avoid peak broadening effects. To achieve an
absorbance of about 0.1 to 0.2 OD at 280 nm with various column
sizes, use a volume of sample proportional to the total column
volume. For example, a column of 170 ml total volume would require
about 0.5 ml of standard, while a column of 40 ml would require about
0.125 ml. Actual chromatographic separations will depend upon
chromatographic techniques and column efficiency.
Table 2. Recommended Volume of Standard
Column Volume Volume of Standard
100–200 ml 0.5 ml
50–100 ml 0.25 ml
25–50 ml 0.125 ml
Figure 1 shows the results achieved with Bio-Sil ® SEC HPLC
columns. For HPLC size exclusion columns (7.8 x 300 mm), a 20 µl
injection loop will give peaks with absorbance at 280 nm of about 0.75.
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