MacroCap SP, 100 mL

Two step constant flow packing. 

Packing buffer: 0.15 M NaCl

For more information regarding packing methods please read Packing MacroCap media in XK 16/20 columns.

The same method is valid for packing MacroCap SP in XK 16, XK 26 and XK 50 columns.

Column	Maximum operation pressure (bar)
HiScale	20
XK 16	5
XK 26	5
XK 50	3
BPG 100	8
BPG 140	6
BPG 200	6
BPG 300	4
BPG 450	2.5
BioProcess LPLC	6
BioProcess MPLC	20
Chromaflow 400	3
Chromaflow 600	3
Chromaflow 800	3
Chromeflow 1000	3
FineLINE Pilot 35	20
FineLINE 70	20
FineLINE 100	20
FineLINE 200	20
FineLINE 350	20

 

BPG 100
10 or 12 µm nets
Adaptor net 10 µm Code no: 18-1103-05
Adaptor net 12 µm Code no: 18-1103-06
End piece net 10 µm Code no: 18-0251-77
End piece net 12 µm Code no: 18-1104-41
BPG 140
10 or 12 µm nets
Adaptor net 10 µm Code no: 18-1113-03
Adaptor net 12 µm Code no: 18-1113-05
End piece net 10 µm Code no: 18-1113-02
End piece net 12 µm Code no: 18-1113-04
BPG 200
10 or 12 µm nets
Adaptor net 10 µm Code no: 18-0252-76
Adaptor net 12 µm Code no: 18-1104-42
End piece net 10 µm Code no: 18-0252-77
End piece net 12 µm Code no: 18-1104-43
BPG 300
10 or 12 µm nets
Adaptor net 10 µm Code no: 18-1012-55
Adaptor net 12 µm Code no: 18-1104-44
End piece net 10 µm Code no: 18-1012-35
End piece net 12 µm Code no: 18-1104-45

 

Packing MacroCap SP in BPG columns

MacroCap SP is packed in BPG columns with conventional flow packing by pumping packing buffer through the chromatographic bed at constant flow rate.

Medium preparation
MacroCap SP is supplied in a solution of 20% ethanol with 0.2 M sodium acetate. Packing using this buffer is not recommended. Instead, decant the ethanol solution from the containers and replace with 0.1 M NaCl in water. Resuspend the slurry and let the medium settle by gravity. For optimal results, carefully remove the supernatant and replace with 0.1 M NaCl in water.

Column preparation
The packing pump should be as pulsation-free as possible. An excenter screw pump is normally the best choice. Level the column with a spirit level. A pressure relief valve should be used for safety reasons and is especially helpful against pressure spikes. Position this valve on the pump outlet and a pressure gauge on the column inlet. Mount a 6-mm 4-port 2-way valve on top of the pressure gauge. Connect the column to the system using 6 mm tubing.

Packing
MacroCap SP is compressible. The required gravity settled bed volume is 1.18 times the targeted packed column volume (the compression factor equals 1.18). To pack a 20-cm bed, the gravity settled bed height in the column should be 23.6 cm.

1. Make sure that no air is trapped under the bottom net by carefully pumping packing buffer through it from below. Leave about 2 cm of buffer in the column. Excess buffer and remaining air can be removed by running a hose connected to the suction side of a pump over the net.

2. Carefully mix the packing buffer and medium to form a homogeneous slurry. The slurry concentration should be 60-65 % (gravity settled bed volume/slurry volume = 0.6 - 0.65). Note that the available height in a 500 mm long column is only 40 cm. For packing beds higher than 20 cm, use a longer column tube; 750 and 950 mm tubes are available.

3. Pour the slurry into the column. Wait until there is about 1 cm clear liquid on top of the slurry. Insert the adapter and lower it to the surface of the slurry. Make sure no air is trapped in the column by allowing some liquid to pass by the O-ring.

4. Seal the adapter O-ring and lower the adapter a little into the slurry, enough to fill the adapter inlet with packing buffer and to remove air trapped in the adapter net. Make sure the top valve is completely primed with buffer. Connect the pump outlet hose to the valve and a hose from the valve to waste.

5. Start pumping at 170 cm/h for 20 cm bed height (or 100 cm/h for 30 cm) and fill the tubing with liquid. Once the tubing is primed, shift the top valve in order to direct the flow to the column and immediately open the bottom valve to waste. Keep the flow running until the bed height is stable and then at least 5 more minutes. Register the compressed bed height and calculate the packed bed height which should be 3 mm below the former. Do not exceed the operating pressure limit of 3 bar for the medium, a typical value should be around 1 bar.

6. Stop the pump and close the bottom valve. To avoid bed expansion, immediately shift the top valve to waste, loosen the adapter O-ring and lower the adapter down to 2 cm above the bed. Seal the adapter O-ring and lower the adapter firmly but gently down to the predetermined packed bed height. The packing buffer will flush the adapter inlet.

7. Remove any trapped air by pumping liquid from the bottom (after the inlet tubing and the bottom valve have been properly filled).



Fig. 1. Pressure/flow curve of a packed bed in BPG 300 at two different bed heights.


Evaluation of column packing
The efficiency of the packed bed should be evaluated at regular intervals during the working life of the column. Check the quality of the packing directly after packing, prior to re-use, and when separation performance is seen to deteriorate. The best method of expressing the efficiency of a packed column is in terms of height equivalent to a theoretical plate, HETP, and asymmetry factor, As. These values are easily determined by applying a sample such as 1% acetone solution to the column using 0.1 M NaCl in water as eluent. Sodium chloride can also be used as a test substance. Use a concentration of 1 M NaCl in water with 0.25 M NaCl in water as eluent.

The calculated HETP will vary depending on the test conditions and should therefore be used as a reference value only. Keep conditions and equipment constant so that results are comparable. Changes in sample composition, sample volume, eluent, nominal fluid velocity (cm/h), liquid pathway, temperature, etc., will influence the results. If an acceptance limit is defined in relation to column performance, the column plate number can be used as part of the acceptance criteria for column use.

To avoid dilution of the sample, apply it as close to the column inlet as possible. Also, place the UV or conductivity meter as close to the column outlet as possible.

Conditions
Sample volume:	1% of the bed volume
Sample conc.:	2% v/v acetone in 0.1 M NaCl
Fluid velocity:	30 cm/h
UV:	280 nm

Calculate HETP and A_s from the UV curve (or conductivity curve if NaCl is used as sample) as follows:
HETP = L/N
N = 5.54(V_e /W_h )²
where L = Bed height (cm)
N = Number of theoretical plates
V_e = Peak elution distance
W_h = Peak width at half peak height
V_e and Wh are in the same units.

To facilitate comparison of column performance, the concept of reduced plate height is often used. Reduced plate height is calculated as:
h = HETP/d_50v
where d_50v is the mean diameter of the bead, using the same unit as for HETP.
As a guideline, a value of h<3 is normally well acceptable at the optimal test conditions presented above.

The peak should be symmetrical and the asymmetry factor as close as possible to 1 (values between 0.8 and 1.5 are usually acceptable). A change in the shape of the peak is usually the first indication of bed deterioration due to use. Peak asymmetry factor is calculated as:
A_s = b/a

where	a = 1st half peak width at 10% of peak height
	b = 2nd half peak width at 10% of peak height.

Fig. 2. An UV trace for acetone in a typical test chromatogram in which the HETP and A_s values are calculated.

One step constant flow packing.

Packing buffer: 0.1 M NaCl

For more information concerning packing MacroCap in BPG columns please read Packing MacroCap media in BPG columns.

The same method is valid for packing MacroCap SP in BPG 100, BPG 140, BPG 200 and BPG 300.

Recommended flow velocities in the table below are for packing the column. 

Column	Compression factor	Slurry concentration (%)	Bed height	Flow velocity (cm/h) Step 1	Flow velocity (cm/h) Step 2
XK 16	1.18	60 - 65	10 - 30	60	150
XK 26	1.18	60 - 65	10 - 30	55	140
XK 50	1.18	60 - 65	10 - 29	50	135

 

Column evaluation
The efficiency of a column depends on how well it is packed. A poorly packed column gives rise to uneven flow, resulting in zone broadening and reduced resolution. It is thus important to have a method by which the column can be tested before it is put into operation. Such a method should be simple, quantitative and should not introduce contaminating materials. It is also an advantage if the same method can be used to monitor column performance over its working life, so that it is easy to determine when the medium should be re-packed or replaced.

Avoid methods that use colored compounds such as Blue Dextran. They do not meet the above criteria and cannot be used with ion exchange and affinity chromatography media.

Experience has shown that the best method of expressing the efficiency of a column is in terms of the height equivalent to a theoretical plate, HETP, reduced plate number, h, and the peak asymmetry factor, As. These values can be determined easily by applying a NaCl or acetone solution, to the column (see below).

It is important that the column is properly equilibrated ( >2 column volumes) before evaluating the packing. Ideally, run three test runs to see whether the values are stable. If an initially poor result improves during a later test, the reason can be that the column was not properly equilibrated. To check that the bed is stable, run the column at 70% packing pressure for 20 hours and test it again.

Note that pressure spikes may cause poor packing (cracking). If this happens, fit an air trap and a pressure relief valve between the pump and column. Locate the pressure relief valve between the air trap and the column.

Choice of test sample for columns
The most appropriate material for column testing is, of course, the sample that is to be run in the application, but this is not always practical or economical. As an alternative, a solution of either NaCl or acetone will give a good indication of the column packing quality. The eluate is monitored by measuring conductivity or UV absorption, and the resulting elution profile is used to calculate the HETP value.

The advantages of using NaCl are that it is readily available and can be used safely to test all columns. One disadvantage is that NaCl may interact with the medium matrix, especially ion exchanger matrices, and thus give erroneous results.

Acetone, in contrast, does not interact with the matrix and is detected by UV absorption at 280 nm. Alternatively, you can increase the running buffer concentration 10-fold and use it as test solution.

The figure below shows a UV trace for acetone in a typical BPG column application and gives calculated HETP and As values.

HETP calculation
The sample volume should be approximately 1% of the total bed volume and the concentration 1.0% v/v NaCl, or equivalent when using stronger buffer. Alternatively, use 1.0% v/v acetone. The flow velocity should be between 10 and 30 cm/h depending on the bead size of the chromatography medium. The high flow velocity could be used for small beads whereas large beads only allow low flow velocity. To avoid diluting the sample, apply it as close to the column inlet as possible. If an airtrap is included in the system, by-pass it during sample application to avoid back-mixing. Calculate the HETP value from the conductivity (or UV) curve as follows:

HETP, in its simplest terms, is expressed as:
HETP = L/N
where,
L = Bed height (cm)
N = Number of theoretical plates.

N is defined by the equation:
N = 5.54 (Ve /Wh)2
where,
Ve = Elution volume (ml)
Wh = Peak width at half height (ml)
Ve is measured as the volume passed through the column to the peak maximum.
Wh is measured as the peak width at half-peak height.
From the example in the figure, the HETP value can be calculated from the chromatogram as follows:

	Ve (ml)	Wh (ml)	N	N/m	HETP cm
Acetone	18800	900	2417	4203	0.024

Well-packed columns have low HETP values. However, it is only possible to compare columns that have been packed with the same type of media and that have been tested under identical conditions.

As a general rule-of-thumb, a good HETP value is approximately two to four times the mean bead diameter of the medium in question, provided that the sample does not interact with the medium.

In practice, the correlation between HETP and column performance can only be assessed by the column operator. Once this has been established, a standard can be set to judge the acceptability of a column packing.

For example, the column operator may know from experience that a column packed with Sephadex G-25 gel filtration medium with HETP values above 0.05 cm does not give the required separation. Consequently, the operator will set this value as the maximum permissible i.e. the minimum acceptable quality.

Reduced plate number
Definition of reduced plate number: h = HETP/dp
h = reduced plate number.
HETP = above described height equivalent to a theoretical plate.
dp = mean particle diameter of the chromatography medium beads.

The reduced plate number should be in the range of 2-4 times the mean particle diameter of the chromatography medium beads.

Peak asymmetry factor calculation
The peak asymmetry factor should be as close as possible to 1, and the shape of the peak should be as symmetrical as possible. This is usually the case for gel filtration media, but for certain ion exchange and affinity media, the shape may be asymmetrical due to interaction with the media. A change in peak shape is usually the first indication of column deterioration.

The peak asymmetry factor, As, is calculated from the graph above:
As = b/a
where,
a = distance from peak apex to 10% of the peak height on the ascending side of the peak
b = distance from peak apex to 10% of the peak height on the descending part of the peak

Note: Measuring HETP, h and As values is the best way to judge the condition of the packed column. A packed column can look good, but still need repacking for optimal performance. Always check the column after packing and regularly between runs to ensure best performance.

Recommended flow velocity intervals in the table below are for packing the column. The intervals are given in reverse order to indicate that the high flow velocity can be used for shorter beds whereas higher beds only allow lower flow velocity, due to the higher pressure drop across the bed.

Column	Compression factor	Slurry concentration (%)	Bed height (cm)	Flow velocity (cm/h)
BPG 100	1.18	60 - 65	10 - 30	250 - 100
BPG 140	1.18	60 - 65	10 - 30	250 - 100
BPG 200	1.18	60 - 65	10 - 30	250 - 100
BPG 300	1.18	60 - 65	10 - 30	250 - 100

Packing MacroCap media in XK 16/20 columns

Materials needed
MacroCap SP
Glass filter funnel
Small spoon or plastic spatula
Filter flask
Measuring cylinder or breaker
20% ethanol
0.15 M NaCl in 20% ethanol

Washing the medium
Equilibrate all materials to room temperature. Mount the glass filter funnel onto the filter flask. Pour the medium into the funnel and wash. Wash with at least 4 ml 0.15 M NaCl per ml medium.

Preparing the medium suspension
The slurry concentration should be 60% in 0.15 M NaCl, measured in a measuring cylinder after settling overnight or after centrifugation at 3000 rpm for 3 min. Wait 5 min after centrifugation before reading.

Equipment needed
Peristaltic Pump, P-1 or High Precision Pump P-500 or P-900.

An ÄKTAexplorer^TM system can be used for packing the column. The inline filter (system filter) unit should be removed due to the high flow velocity used in column packing. Removing the filter decreases the system backpressure.

The columns should be packed without a slurry reservoir as it is important to lock the medium bed quickly when packing is complete.
XK 16/20 is used for 10-cm bed heights.
XK 16/40 with two flow adapters is used for 15-cm bed heights. If one flow adapter and end piece is used, a bed height of 20 cm can be obtained.

Assembling the column
Details of the column parts can be found in the instructions supplied with the column. Before packing, ensure that all parts, particularly the nets, net fasteners, and glass tube are clean and intact.

1. Wet the bottom filter with 20% ethanol with a syringe mounted on the outlet tubing. After the filter is wetted, mount a stop screw on the outlet.

2. Mount the bottom piece in the column tube.

3. Wet the adapter filter with 20% ethanol with a syringe mounted on the inlet tubing. After the filter is wetted, mount a stop screw on the inlet.

4. Keep the adapter in a breaker filled with 20% ethanol.

5. Mount the column vertically on a stand.

6. Fill the glass tube with the 60% medium slurry up to the upper edge of the glass tube.

7. Mount the adapter in the upper part of the glass tube and make sure that no air is trapped below the filter.

8. Tighten the sealing ring and connect the column inlet to the system outlet.


Packing procedure
To pack the column, use 0.15 M NaCl in 20% ethanol and proceed as follows:

1. Pack the column at 60 cm/h (2.0 ml/min, XK 16) for 30 min or until the bed height and the backpressure are constant. Stop the pump and mount a stop screw on the outlet.

2. Increase the flow rate to 150 cm/h (5.0 ml/min) and continue packing for 5 min. Adjust the adapter quickly down to the medium surface and then a further 5 mm into the medium bed. Lock the adapter at that level.

Note: Do not exceed the operating pressure limit for the medium (3.0 bar not including system effect.)


Troubleshooting
If the column does not fulfill the criteria with respect to h and A_s, described in selection "Evaluation of column packing", empty the column, and repack according to the table, and perform a new test.

Situation	Action
h > 3 and As < 0.8	Decrease the flow rate in step 1 of the packing procedure from 2.0 to 1.5 ml/min. Decrease the flow rate in step 2 from 5.0 to 4.0 ml/min.
h > 3 and As > 1.5	Decrease the flow rate in step 1 from 2.0 to 1.5 ml/min. Increase the flow rate in step 2 from 5.0 to 5.5 ml/min.
h < 3 and As < 0.8	Decrease the flow rate in step 2 from 5.0 to 4.0 ml/min.
h < 3 and As > 1.5	Decrease the flow rate in step 1 from 2.0 to 1.5 ml/min. Increase the flow rate in step 2 from 5.0 to 5.5 ml/min.

XK 16/40
10 µm nets (5 pieces/pack) Code no: 18-8761-01
XK 26/40
10 µm nets (5 pieces/pack) Code no: 18-8760-01
XK 50/30
10 µm nets (5 pieces/pack) Code no: 18-8759-01