Prepacked columns from Cytiva will ensure reproducible results and the highest performance.
Use small prepacked columns for chromatography media scouting and method optimization and to increase efﬁciency in method development.
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 A5.1).
Figure A5.1.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 (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 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:
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:
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 A5.2.
Figure A5.2.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:
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.
Tricorn, XK, and HiScale® columns are fully compatible with the high ﬂow rates allowed by with modern media, and a broad range of column dimensions are available (Table A5.1).
In most cases the binding capacity of the medium and the amount of sample to be puriﬁed will determine the column size required. Also, Empty Disposable PD-10 Columns are available for single-use applications using gravity ﬂow.
After puriﬁcation, the medium should be regenerated as follows:
When an increase in backpressure is seen, the medium should be cleaned. In some applications, substances like denatured proteins or lipids do not elute in the regeneration procedure.
To remove precipitated or denatured proteins:
To remove strongly bound hydrophobic proteins, lipoproteins and lipids:
After treatment, wash with at least 5 column volumes of binding buffer.
Reversed ﬂow can improve the efﬁciency of the cleaning-in-place procedure. After cleaning, store in 20% ethanol.
Washing with 70% ethanol will increase backpressure. Use a lower ﬂow rate when cleaning with 70% ethanol.
All MabSelect media can be cleaned using the following procedures:
To remove precipitated or denatured substances:
MabSelect SuRe and MabSelect SuRe LX are alkali-tolerant, allowing the use of more concentrated solutions of sodium hydroxide:
To remove strongly bound hydrophobic proteins, lipoproteins, and lipids:
Washing with 70% ethanol and 30% 2-propanol will increase backpressure. Use a lower ﬂow rate when cleaning with 70% ethanol or 30% 2-propanol.