Prepacked columns from Cytiva will ensure reproducible results and the highest performance.
Use small prepacked columns for media screening and method optimization to increase efficiency in method development, for example, HiTrap™ HIC Selection Kit, RESOURCE™ HIC Test Kit and RESOURCE™ RPC columns.
Efficient column packing is essential for a good separation, especially when using gradient elution.
A poorly packed column gives rise to poor and uneven flow, peak broadening, and loss of resolution.
If column packing is required, the following guidelines will apply at all scales of operation:
HIC media can be packed in either Tricorn or XK columns available from Cytiva. SOURCE™ RPC can be packed in Tricorn or HR columns also available from Cytiva. A step-by-step demonstration of column packing can be seen in “Column Packing – The Movie”, available in CD format.
Note that HIC media from Cytiva are supplied ready to use. Decanting of fines that could clog the column is unnecessary
Avoid using magnetic stirrers since they may damage the matrix.
When slurry volume is greater than the total volume of the column, connect a second glass column to act as a reservoir (see 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 the maximum operating pressure of the medium or column.
Do not exceed 75% of the packing flow rate during any purification.
The medium must be thoroughly washed to remove the storage solution, usually 20% ethanol. Residual ethanol may interfere with subsequent procedures.
Many 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.
Tricorn and XK columns are fully compatible with the high flow rates achievable with modern media and a broad range of column dimensions are available. Columns most suitable for packing HIC media are listed under the column packing section for each HIC medium (Chapter 3). In most cases the binding capacity of the medium and the amount of sample to be purified will determine the column size required. For a complete listing, refer to the Cytiva life sciences catalog.
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. Since column efficiency is related to the peak broadening that can occur on a column, it 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 HIC, 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 89.
Figure 89. 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 0.018–0.027 cm.
The symmetry factor (As) is expressed as:
|As =|| b
a = 1st half peak width at 10% of peak height
b = 2nd half peak width at 10% of peak height
As should be as close as possible to 1. A reasonable As value for a short column when used for HIC or RPC is 0.80–1.80.
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 two column volumes 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.