Instructions for Special Packing Material for Phosphoramidite

Instructions for Special Packing Material for Phosphoramidite

Product Introduction

The special packing material for phosphoramidite is made of high-purity silica gel which is independently developed and produced by Biosepur. With a special pore optimization technique and unique surface treatment technique, this packing material ensures uniform pore size and excellent binding capacity. Biosepur further adopts unique silica gel bonding and end-capping techniques to ensure its excellent selectivity and chemical stability. The special packing material for phosphoramidite can be used for the reversed-phase purification process of a variety of phosphoramidite monomers. The bonding process of the packing material enables it to meet all the performance requirements for the purification of phosphoramidite monomer, especially the recovery and the specific impurity separation performance.

Product parameters

Bonded phase	C18	C18
Particle size μm	30	50
Pore size Å	100	100
Carbon binding capacity %	17	17
Specific surface area m2/g
pH range	2-9	2-9
Pressure resistance MPa	5	5
Common mobile phase	Water, acetonitrile	Water, acetonitrile
Common flow rate range mL/min (50 mm DAC; for other column diameters, the flow rate is proportional to the cross-sectional area)	50-80	50-80

Precautions for use

• The special packing material for phosphoramidite can be used in a wide pH range due to its superior chemical stability. However, long-term use in the condition of strong alkali and high salt will reduce its service life.
• In addition to the salt concentration and pH of the mobile phase, the service life of the packing material is also related to the residual contamination on the packing material by strong adsorbent substances or macromolecules in the sample or solvent. The packing material should be cleaned in time after use to maintain its service life; otherwise, if the sample or solvent contains too many raw materials and oxidation and hydrolysis impurities, dead adsorption and other contaminants will be generated on the packing materials, resulting in a decrease of the service life. Therefore, we adopt the method of in-column/out-of-column treatment for packing material regeneration to meet the requirements for the service life.
• For samples with more impurities, pretreatment such as filtration may be carried out in advance or protective columns can be used.

Storage of packing material

• Unused packing material: Store directly in factory containers and avoid high temperature and humid conditions for a shelf life of 5 years.
• Used packing material: After the preparation process is completed, the column shall be cleaned according to specific cleaning method: The residual buffer salt on the column must first be washed off with low-concentration organic phase water system (acetonitrile/water is recommended), and then flushed with a high-concentration organic phase. After the cleaning, the packing material can be stored wet or dry in the column. If the used silica gel packing material is to be stored wet in a solvent, an anhydrous and neutral organic solvent is recommended; for dry storage, use an organic solvent such as acetonitrile to replace the packing material, then release the packing material from the column, dry it in an oven (at a temperature not higher than 90 ℃), and then store the packing material in a sealed container at room temperature.

Packing and evaluation of preparative MP column

1. Packing method

• Determination of packing capacity:

Packing capacity = density of packing material × column volume (Note: It is recommended to weigh 0.9-0.95 times the theoretical amount of packing material to ensure space for adding samples at the top of the column bed.)

• Selection of appropriate mixing solvent:

To ensure the stability of phosphoramidite, we generally use acetonitrile for slurry packing of special packing material for phosphoramidite. The acetonitrile will be mixed with the packing material at a ratio of 1.4 ml/1g or so before packing.

• Mixing:

Select an appropriate container according to the calculated volume (For 300 g of packing material, 1 L beaker or other container shall be used for mixing. If the amount of packing material is up to 5 kg, a 10 L plastic barrel can be used for mixing, and so on); pour 50% of the estimated amount of mixing solvent into the container, and add the weighed packing material, and then slowly add the remaining solvent to the packing material while stirring (do not pour all the solvent at one time), stir for 5-10 minutes to disperse the packing material evenly in the solvent, and finally the packing material will become a more viscous fluid, which can be packed directly.溶剂中均匀分散，最终填料会成为较为粘稠的流体状态，此时可以直接进行装填。

• Packing:
• Wash the column tube with acetonitrile and then blow it dry with an air gun to ensure there is no moisture residue. The lower cover plate is cleaned with acetonitrile and then attached to the bottom of the column using a hydraulic trolley, and tightened by connectors;
• Pour the slurry into the column tube. At this time, you need to place an identical container under the outlet to recover the slurry that flows out the outlet at the bottom of the column;
• When 90% of the packing material is added to the column tube, the tube may seem to be fully packed. Leave it to stand for 20 minutes to allow the whole column bed to be fully settled. Then add the remaining slurry to the column tube and wash;
• Acetonitrile should be used to circulate 3-5 column volumes after the column is packed for the first time to ensure the bed is intact and fault-free.
• Discharging
• Lift the hydraulic trolley jack to the lower cover plate to hold it against the lower cover plate, remove the connectors, and slowly lower the hydraulic trolley jack to release the lower cover plate;
• At this time, the packing material will flow out slowly from the bottom of the column. Use a container to collect it. An infusion pump can be used to pump pure acetonitrile from the upper end to speed up the discharging;
• After the packing material is completely discharged, use acetonitrile to clean the column tube, or if possible, use an air gun to dry it. Bake the packing material or store it in pure acetonitrile. DO NOT use pure water for storage.

2. Evaluation of column performance

After packing, the column packing effect is generally evaluated using LC system, including the confirmation of the number of theoretical plates (N) and the peak shape. If the column efficiency evaluation results do not meet expectations, the packing process can be adjusted appropriately to seek the best effect.

Sample and sample loading

The special packing material for phosphoramidite from Biosepur carries out special treatment for phosphoramidite monomers to meet the separation requirements of a larger sample loading capacity. The theoretical maximum sample loading capacity of crude product is 10% of the mass of packing material. Due to the strong alkalinity of some phosphoramidite monomers, it is necessary to use buffer salts (such as sodium bicarbonate, etc.) for adjustment before injection to maintain the pH of the sample not more than 9.

For the preparative MP column packed with the special packing material for phosphoramidite, it is necessary to load the sample in the reversed-phase mode, that is, to dissolve the sample and load it using the pump. Generally, 1 g of sample can be dissolved with 2 mL of pure acetonitrile. During the dissolution of the sample, the column can be equilibrated with mobile phase of acetonitrile and water. To stabilize the sample, buffer salts (such as triethylamine, diisopropylethylamine, etc.) will be added to the mobile phase to stabilize the pH at around 8.5.

Cleaning of packing material

After a long period of use, some strongly retained substances in the samples may accumulate and adsorb on the surface of the packing material, which is difficult to elute and will seriously affect the performance and subsequent use effect of the packing material. To solve this problem, we tested the packing material cleaning with phosphoramidite-related samples, and finally, we obtained the cleaning method (which allows in-line cleaning as well as transferring to a vessel):

Weigh 50 g of packing material, measure 100 mL of methanol, and add 1 mL glacial acetic acid (17.5 mol/L) to the methanol. Pour the methanol solution into the packing material and heat it up to 40 ℃, stirring to clean it for 30 minutes, and then wash it with 100 mL of methanol, drain it with a sand–core funnel, dry it in an oven at 100 ℃, and pack into a FLASH column for testing. For packing material with a larger mass, a scaled-up amount of cleaning agent can be used for cleaning.

The internal cleaning of the column can also follow the above method, but the amount of cleaning agent should be scaled up 3-4 times to ensure the cleaning effect and subsequent performance.

Packaging and transportation

According to the needs of most customers, Tianjin Biosepur products are available in two standard packages of 5 Kg and 25 Kg; if you need other specifications, please contact our sales representatives.

After-sales service

The application and purification processes support department of Tianjin Biosepur will provide you with professional guidance and services. You can also call our after-sales service number 400-800-6028, and our customer service representatives and professionals are always available for your consultation.