BESTCHROM Bestdex LH-20 Dextran Resin Instruction Manual

: June 9, 2024
: BESTCHROM

Table of Contents

Introduction
Technical characteristics
Method of chromatographic
Influencing factors of separation effect
Cleaning-in-place (CIP)
Sterilization
Storage
Disposal and Recycling
Order information
References
Read User Manual Online (PDF format)
Download This Manual (PDF format)

Bestdex LH-20
Dextran Resin
Instruction for usehttp://www.bestchrom.com/

Introduction

Bestdex LH-20 is modified with hydroxypropyl on the basis of dextran gel Bestdex G-25.The introduction of hydroxypropyl makes the resin both hydrophilic and lipophilic. Organic solvents can be used as mobile phase. It is suitable for separation and purification of active ingredients in traditional Chinese medicine as well as fine purification of antibiotics and chemical drugs. The resin combines the characteristics of gel filtration, distribution chromatography, and adsorption chromatography to separate molecules with very similar structures.

Technical characteristics

Appearance	White or off-white powdery solid
Matrix	Dextran gel
Particle size(dry)+	30~120μm
Average particle size(dry)++	75μm
Average particle size(wet)	Large difference in different solutions
Exclusion limit	4~5KD
Max. Linear rate	700cm/h
Max. Pressure	0.3MPa
Chemical stability	Stable in water and most organic solvents. It’s unstable

below pH2, and it’s also unstable to strong oxidants.
pH Stability| 2~13
Working temperature| 2~40℃
Recommended Linear rate| 60cm/h

+Particle size is normally distributed, and particles within this range account for more than 95% of the total.
++ There will be differences in different solutions, this data is the particle size in PBS
List of swelling ratio of Bestdex LH-20 in different solvents:

AWIRMII| welling rati| Solvent
Propanol| Swellin. ratio
3.7∼4.0
---|---|---|---
Dnnethyl sulfoxide| 4.4∼4.6
Pyridine| 4.2∼4.4| Ethanol| 3.6∼3.9
Water| 4.0∼4.4| Isobutanol| 3.6∼3.9
Dimethylformamide| 4.0∼4.4| Formamide| 3.6∼3.9
Methanol| 3.9∼4.3| Methylene chloride| 3.6∼3.9
Dichloroethylene| 3.8∼4.1| Butanol| 3.5∼3.8
Chloroform| 3.8∼4.1| Isopropanol| 3.3∼3.6
Tetrahydrofuran| 3.3∼3.6| Acetone| 2.4∼2.6
Acetonitrile| 2.2∼2.4| /| /

Note：The swelling ratio is the volume(mL)per gram of dry powder after swelling.

Method of chromatographic

The resin is provided as a dry powder and must be swollen before use.
3.1 Swelling

Calculate the required amount of Bestdex LH-20 dry powder according to the volume of the chromatography column.
Dry powder(g) =(CV×1.15)÷ swelling ratio.
Pour the resin into the corresponding solvent of 5 times the weight of dry powder and stir it a little, swelling generally takes more than 4h (note that during the swelling process, the use of magnetic stirrer will cause the resin particles to rupture so do not use a magnetic stirrer to stir). After swelling at normal temperature, it is best to degassing under negative pressure.
After swelling is completed (the swelling at high temperature needs to be cooled to room temperature), remove part of the supernatant to make the volume of the sedimentation gel account for 50% to 75% of the total volume, and mix well for use.

3.2 Column packing

Note: Since Bestdex LH-20 is often used in organic solvents, please make sure that the chromatography column you use can tolerate the corresponding organic solvents. High concentration of organic solvent will cause the laboratory BXK column shell to rupture. During the packing process, be careful not to let the organic solvent contact the laboratory BXK column shell.
When using dichloromethane or chloroform to pack the column, the resin will float on the surface of the liquid. Use double column head to load columns from bottom to top.
Use a liquid consistent with the swelling fluid as the packing buffer.
Take a cleaned BXK column (BXK series columns with diameters ranging from 1cm to 30cm can satisfy different scale chromatography applications). Purge the bubbles trapped at the end-piece net by draining some packing solution through the column outlet. Leave about 1cm water at the bottom of the column and close the bottom outlet. Adjust the column so that it is perpendicular to the ground.
Slowly pour the slurry into the column at one time (use a packing reservoir if necessary). Do not bring any air bubbles into the column.
Packing reservoir: Empty glasstube with same diameter as the BXK column.
Fill the remainder of the column with packing solution. Connect the packing reservoir to the chromatography system, open the flow rate, drain the bubbles in the hose, close the flow rate, and tighten the top cover of the packing reservoir.
Note: This operation is only applicable to BXK 50 and below chromatographic columns.
After pouring, stir well again with stirrer, and then wash the resin particles on the inner wall of the column from top to bottom with the packing solution, and let the resin settle naturally until there is about 1cm of clarifying solution on the suspension.Mount the adapter and connect the adapter to the chromatography system or peristaltic pump.Lower the adapter to descend to contact with the clarifying solution and tighten the sealing ring after it is fully immersed in the clarifying solution.With the outlet of the top piece is opened, slowly move the adapter down until all bubbles are drained.
Note: This operation is only applicable to BXK 100 and above columns.Flushing the inner wall reduces the resin particles sticking between the seal ring and the column wall, avoiding the risk of leakage.
- Set the packing flow rate. Due to the greater hardness of Bestdex LH-20 gel, the flow rate is almost proportional to the pressure. The pressure within 3Bar will not cause damage to the resin.
  The packing should be completed at the largest possible flow rate.
- Open the bottom end of the column and start the peristaltic pump or chromatography system according to the flow rate set above. The pressure in the column is set to be less than the maximum pressure of the chromatography column. If the pressure is exceeded during column packing, the flow rate needs to be reduced appropriately.
- After the settlement of the glue suspension needs to be maintained for more than 30 minutes, mark the position of the glue surface, and then stop the pump.
- Remove the packing reservoir (if any), lower the adapter to about 0.5cm above the resin surface, and continue to press the column using the above flow rate until the bed is completely consolidated, marking the consolidated bed height.
- Stop the pump, open the outlet of the top piece, close the outlet of the bottom piece, loosen the seal ring slightly, press the adapter to about 0.3cm below the marked position, tighten the seal ring, close the outlet, and complete the column packing.

3.3 Evaluation of Packing

Test the column efficiency to check the quality of packing.The tests are required after the column packing, during the column working life and when the separation and purification performance is deteriorated. The method of the expressing the efficiency of a packed column is in terms of the height equiv valent to a the eoretical plat te(HETP) and d the asymme etry factor(As).
Acetone or prepared ac NaCl can be used as sam ccording to th he following t mple for the te table.

Acetone method NaCI method

Method for measuring HETP and As:
According the UV curve or the conductivity curve to calculate the column efficiency(HETP), and the asymmetry (As):
HETP=L/N
N=5.54(VR/W02
Note: VR = retention volume
Wh = half-peak width
L = column height
N = the number of theoretical plates
(The units of VR and Wh should be the same)
As=b/a
Note:
a= 1st half peak width at 10% of peak height
b= 2nd half peak width at 10% of peak height
Evaluation the column packing
As a guideline, if the value of HETP is less than 3 times the average particle size(d50)of the resin and the As is between 0.81.8, it is very efficiint. The unsatisfactory results should be analyzed and the column should be repacked .

3.4 Chromatographic method

Buffer selection: Bestdex LH-20 can be run in various aqueous or organic phases. The mobile phase is selected according to the nature of the species being separated and the principle of distribution chromatography. A mixed solution of ethyl acetate, methanol, and water is often used. Adjust the ratio of each substance in the mobile phase according to the peak time of the target.
Flow rate: Take the column height of 80cm as an example, the generally recommended flow rate is about 30 — 90cm / h, the greater the column height, the slower the flow rate.
Sample preparation: In order to prevent blocking of the column, the sample needs to be filtered by microporous membrane of 0.45pm before loading.
Equilibration: The chromatographic column is washed with the equilibrium buffer solution. The pH and conductivity of the buffer solution to be exported are consistent with that of the buffer solution before entering the chromatographic column, which means that the chromatographic column is balanced. Generally, 2-5CV are required. If the solvent is changed, note the swelling properties of the medium in the new solvent and adjust the position of the adapter accordingly.
Sampling: The sample is loaded onto the chromatography column through the loading ring and other devices of the chromatography system. The volume of the loading sample is different according to the molecular size of the object and impurities or the separation method.The loading volume is usually 1-2% of the column volume.
Rinse: According to the composition of the sample, set the elution flow rate (1-10cm /h is recommended) for elution, and the smaller the flow rate, the better the separation effect.
Regeneration: 2-3CV volumes of the chromatographic column were washed with a balanced buffer solution.
Rebalancing: After rinsing with equilibration buffer, the second sample can be loaded and repeated.

Influencing factors of separation effect

The separation effect of Bestdex LH-20 gel resin is affected by various principles:

Gel filtration: If there is no interaction between the target and the resin, the separation is based on the principle of molecular sieve. The choice of buffer mainly considers the solubility of the target. The influencing factors of solubility refer to the influencing factors of gel filtration. Factors such as column efficiency, flow rate and sample loading were considered.
Distribution chromatography: According to the principle of similar compatibility, the target can be reasonably distributed in the mobile phase and the stationary phase by adjusting the polarity of the mobile phase.
Adsorption: The small amount of ethoxy group and hydroxypropyl group contained in the cross-linking agent can bind the biomolecule through the hydrophobic effect. The hydroxyl group of the hydroxypropyl group can bind the biomolecule through the action of hydrogen bonding. The above two aspects can be considered and adopt corresponding measures if binding occurs.

Cleaning-in-place (CIP)

Bestdex LH-20 series resin may decrease the column efficiency after using for a period of time, and the separation effect becomes poor. The following process can be used for cleaning and regeneration.

Rinse 2CV with distilled water
Flush 1CV with 1M NaC1
Rinse 1CV with 0.2M NaOH
Rinse 4CV with distilled water

Sterilization

The swollen Bestdex LH-20 can be autoclaved at 121℃ for 20 minutes or treated with 0.5M NaOH for 30~60 minutes to reduce the risk of microbial contamination.

Storage

The dry powder Bestdex LH-20 is air-tightly stored in a cool and dry place to prevent moisture absorption. The swollen Bestdex LH-20 should be stored in 20% ethanol and sealed at 2-30℃ after use, in order to prevent ethanol volatilization and microbial growth, it is recommended to replace 20% ethanol every 3 months, and the effect is better when stored at 2-8℃.