Prepacked columns from Cytiva will ensure reproducible results and the highest performance.
Use small prepacked columns for media scouting and method optimization, to increase efficiency in method development e.g. HiTrap IEX Selection Kit.
Efficient column packing is essential for IEX 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:
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 month, 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 IEX media are listed under the column packing section for each IEX medium. In most cases the capacity of the IEX medium and the amount of sample to be purified will determine the column size required. For a complete listing refer to the Cytiva BioDirectory™ or web catalog (https://www.cytivalifesciences.com/en/us/solutions/protein-research) or visit www.tricorncolumns.com for more details on Tricorn columns.
Column packing and efficiency
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 band broadening which can occur on a column, it can be calculated from the expression:
N = 5.54 × (VR/Wh)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 height
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 IEX, 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 92.
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 as used in IEX 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.