Column Packing and Preparation for Hydrophobic Interaction and Reversed Phased Chromatography

Appendix 1, extracted from Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods (PDF), GE Healthcare, 2014

Prepacked columns from GE Healthcare 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:

• When using a binding technique, use short, wide columns (typically 5–15 cm bed height) for rapid purification, even with low linear flow.
• The amount of medium required will depend on the binding capacity of the medium and the amount of sample. The binding capacity of a medium is always significantly influenced by the hydrophobic nature of the sample as well as the medium itself and must be determined empirically. Estimate the amount of medium required to bind the sample of interest and use five times this amount to pack the column. The amount of medium required can be reduced if resolution is satisfactory.
• Once separation parameters have been determined, scale up a purification by increasing the diameter of the column to increase column volume. Avoid increasing the length of the column as this will alter separation conditions.

HIC media can be packed in either Tricorn or XK columns available from GE Healthcare. SOURCE™ RPC can be packed in Tricorn or HR columns also available from GE Healthcare. A step-by-step demonstration of column packing can be seen in “Column Packing – The Movie”, available in CD format.

1. Equilibrate all materials to the temperature at which the separation will be performed.
2. Eliminate air by flushing column end pieces with the recommended buffer. Ensure no air is trapped under the column net. Close column outlet leaving 1–2 cm of buffer in the column.
3. Gently resuspend the medium.

Note that HIC media from GE Healthcare 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.

1. Estimate the amount of slurry (resuspended medium) required on the basis of the recommendations supplied.
2. Pour the required volume of slurry into the column. Pouring down a glass rod held against the wall of the column will minimize the introduction of air bubbles.
3. Immediately fill the column with buffer.
4. Mount the column top piece and connect to a pump.
5. Open the column outlet and set the pump to the desired flow rate.

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.

1. Maintain the packing flow rate for at least 3 column volumes after a constant bed height is obtained. Mark the bed height on the column.

Do not exceed 75% of the packing flow rate during any purification.

1. Stop the pump and close the column outlet. Remove the top piece and carefully fill the rest of the column with buffer to form an upward meniscus at the top.
2. Insert the adaptor into the column at an angle, ensuring that no air is trapped under the net.
3. Slide the adaptor slowly down the column (the outlet of the adaptor should be open) until the mark is reached. Lock the adaptor in position.
4. Connect the column to the pump and begin equilibration. Re-position the adaptor if necessary.

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.

Column selection

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 GE Healthcare life sciences catalog.

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 peak broadening that can occur on a column, it can be calculated from the expression:

N =

5.54

X

(VR)2 Wh

V_R = volume eluted from the start of sample application to the peak maximum
W_h = peak width measured as the width of the recorded peak at half of the peak heightt

H is calculated from the expression:

H =

L N

L = height of packed bed.

Measurements of V_R and w_h 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

where

a = 1st half peak width at 10% of peak height

b = 2nd half peak width at 10% of peak height

A_s should be as close as possible to 1. A reasonable A_s 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.