Efﬁcient column packing is essential for AC separation, especially when using gradient elution. A poorly packed column gives rise to poor and uneven ﬂow, band broadening, and loss of resolution. If column packing is required, the following guidelines will apply at all scales of operation:
AC media can be packed in either Tricorn, XK, or HiScale columns available from Cytiva (Figure A3.1).
Figure A3.1.Column packing in progress
Note that AC media from Cytiva are supplied ready to use. Decanting of ﬁnes that could clog the column is unnecessary.
Avoid using magnetic stirrers since they can damage the chromatography matrix.
When slurry volume is greater than the total volume of the column, connect a second glass column to act as a reservoir (Ordering information for details). This ensures that the slurry has a constant diameter during packing, minimizing turbulence and improving column packing conditions.
If the recommended ﬂow rate cannot be obtained, use the maximum ﬂow rate the pump can deliver.
Do not exceed 70% of the packing ﬂow rate during any puriﬁcation.
The medium must be thoroughly washed to remove the storage solution, usually 20% ethanol. Residual ethanol can interfere with subsequent procedures.
Many chromatography media equilibrated with sterile phosphate-buffered saline containing an antimicrobial agent may be stored at 4 °C for up to 1 mo, but always follow the speciﬁc storage instructions supplied with the product.
Column efﬁciency is expressed as the number of theoretical plates per meter chromatography bed (N) or as H (height equivalent to a theoretical plate, HETP), which is the bed length (L) divided by the plate number. Column efﬁciency is related to the band broadening that can occur on a column and can be calculated from the expression:
|N =||5.54||X|| (VR)2
VR = volume eluted from the start of sample application to the peak maximum
Wh = peak width measured as the width of the recorded peak at half of the peak heightt
H is calculated from the expression:
|H =|| L
L = height of packed bed.
Measurements of VR and wh can be made in distance (mm) or volume (mL) but both parameters must be expressed in the same unit.
Column performance should be checked at regular intervals by injecting acetone to determine column efﬁciency (N) and peak symmetry (asymmetry factor, As). Since the observed value for N depends on experimental factors such as ﬂow rate and sample loading, comparisons must be made under identical conditions. In AC, efﬁciency is measured under isocratic conditions by injecting acetone (which does not interact with the medium) and measuring the eluted peak as shown in Figure A3.2.
Figure A3.2.Measurements taken to calculate column efﬁciency.
As a general rule, a good H value is about two to three times the average particle diameter of the medium being packed. For a 90 µm particle, this means an H value of 0.018 to 0.027 cm.
The asymmetry factor (As) is expressed as:
|As =|| b
a = First half peak width at 10% of peak height
b = Second half peak width at 10% of peak height
As should be as close as possible to 1.0. A reasonable As value for a short column as used in AC is 0.80 to 1.80.
An extensive leading edge is usually a sign that the medium is packed too tightly and
extensive tailing is usually a sign that the medium is packed too loosely.
Run at least 2 CV of buffer through a newly packed column to ensure that the medium is equilibrated with start buffer. Use pH monitoring to check the pH of the eluent.
A service for packing of laboratory columns or ﬁlling of 96-well plates is supplied when columns or plates with suitable chromatography media are not available from the standard portfolio. The Custom Products group works in close collaboration with you to deliver packed columns for specialized puriﬁcation requirements.