Application Notes 

Chromatography separation

Chromatography Company

Water Polar.jpg
Water molecule 
Polar molecule, One side that is positively charged and one side that is negatively charged. Water Molecule, has slightly higher negative charge. One side of the water molecule has a weak positive charge while the other side has a weak negative charge. A covalent bond is a chemical bond that forms when atoms share electrons. The oxygen side attracts electrons to a greater degree than the hydrogen side, which draws electrons away from the hydrogen atoms. This leaves each side with a partial charge (positive on the hydrogen side, negative on the oxygen side). Like two bar magnets, whose oppositely charged ends are attracted to one another, two water molecules are attracted in a similar way.  As a polar molecule, water does not interact well with any nonpolar molecule, whose sides carry equal or no electrical charges. Water molecules cannot easily form hydrogen bonds with nonpolar molecules. This means that nonpolar molecules do not dissolve well in polar solvents, such as water, acetic acid, and ethanol.
Ionic Bond.jpg
Ionic bond
The chemical bond formed between two atoms by complete transfer of one or more electrons from one atom to other so as to attain their nearest inert gas configuration by each atom, that is donating or accepting electrons to complete their octet configuration. When the atom loose a electron, it is called ion. Two ions are formed, oppositely charged  -ve ions ( anions) & +ve ion (cations). The bond formed by this kind of combination is known as an ionic bond.
Polar NonPolar Bond.jpg
Covalent bond
Covalent bond forms when valence electrons are shared between atoms. Nonpolar covalent bonds form between two identical with identical affinities for electrons, in which the nuclei of the bonded atoms exert nearly equal force on the shared electrons. A polar covalent bond is a covalent bond in which the electron density is more localized on one end of the bond, with one end is slightly positive, and one end is slightly negative. Electrons are shared equally in a nonpolar molecule, such as fluorine (F2). Electrons are shared unequally in a polar molecule, such as hydrogen fluoride (HF).

Asymmetry

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Tailing Factor

Tailing Factor-LC Process.jpg

N Plates

NPlates-LC Process.jpg

N Plates

NPlates 5.54-LC Process.jpg

Scale Up Calculation

It is recommended to scale up method parameters using simple calculations for flow rate and loading volume.

 

Scale up Flow Rate = Analytical Flow Rate x [( Internal Diameter of Prep Column )²/ ( Internal Diameter of Analytical Column )²]

 

Scale up Loading = Loading Qty on Analytical x [( Internal Diameter of Prep Column )²/ ( Internal Diameter of Analytical Column )²] x [ Bed Length of Prep Column / Length of Analytical Column ]

 

C18 Stationary Phase

Carbon chain attached to Silica. A chain of 18 carbon molecule are bonded with SiO. These C18 interact with compound to assist in adsorbing the sample on the column stationary phase. and help separation. It is important to carefully select good quality, mechanically  and chemically strong stationary phase for purification.  
C18 Ligand SiO.jpg

DAC Column Packing Procedure

How to pack a DAC Column. There are various procedure that are followed to pack a Dynamic Axial Compression Column, more or less similar to each other. We are sharing one of the procedure that can help to understand the how the packing process works. It is also important to understand that use of DAC Column. The DAC Column is used in chromatography process along with Prep or Plant scale HPLC System for large scale purification. The stationary phase such as C18, Silica, Chiral etc. is filled in the column. The Hydraulic is then compressed, that apply axial pressure on stationary phase. The hydraulic piston maintain the continuous pressure and so the constant bed height of stationary phase. A good DAC Column always ensure compact stationary phase bed, without distortion, without channeling, and uniform pressure all through.
 
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Step - I

The first step for DAC Column Packing is to mix the stationary phase and solvent using the slurry tank, provided along with the DAC Column. Handle the stationary phase with care, and ensure that fines are not generated and legands are intact. Keep mixing gently till the mixture become homogeneous. Use the solvent that is recommended for preparing slurry. Ethanol, IPA are also used in general. The quantity of solvent should be 1.5 to 2.5 times depending on the type of packing. 
LCP-Step-2-DAC.png

Step - II

After homogeneous mixing of media and solvent in the slurry mixing tank, the slurry is then transferred using a pumping device, from slurry tank to DAC Column. the Slurry tank and DAC Column are connected with appropriate piping. Ensure static discharge connections. Now the DAC Column is filled with slurry. The piston of DAC Column is sitting in home position. Slurry transfer procedure very important step and should be carried out as per SOP.
LCP-Step-3-DAC.png

Step - III

The slurry tank can be disconnected after slurry transfer. Once the slurry is in the column, bring the piston down gradually. Using the convenience valve available on DAC Column, follow the sequence of opening and closing the valve while the piston is moving in downward direction. The piston will push the solvent out of the column. Piston will automatically stop once the set packing pressure is reached. The standard procedure should be carefully followed while performing this step.
LCP-Step-4-DAC.png

Step - IV

Once the DAC Column is packed, it should be kept idle for 24 Hrs for the slurry to settle. This can help achieve good compact uniform bed. The Hydraulic will apply continuous pressure on the stationary phase, ensuring that bed is always compact without any voids. It is recommended to check the column for N Plates and Asymmetry using evaluation test sample and standard test method. The Plates, Tailing factor should be in range, which ensure that column bed is uniform without any channel and void. The DAC Column is now ready to get loaded with sample. 

Ion Exchange Chromatography

 
Ion Exchange +.png
A mixture of positive and negative charged constituents are loaded on to the Ion Exchange media, that is either positively charges beads or negatively charged beads. Oppositely charged constituents bind to the charged beads. Unbounded constituents are washed out of the column. The bonded constituents then elute out of the column based on the mobile phase charge.
Ion Exchange -.png
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