Column Chromatography

PRODUCTS:SILICA GEL

ALUMINIUM OXIDE

What is Chromatography?Chromatography has been developed into a new method of

separation of mixture of compounds mainly when they are available in small quantities.

This method is very useful when the components of a mixture have almost the same physical and chemical properties and hence can’t be separated by other usual methods of separations.

The term chromatography means writing in colour (Chroma = Colour & Graphy = To write).

Types of Chromatography

• Paper Chromatography

• Gas Chromatography

• Thin Layer Chromatography

• Solid - Liquid Chromatography (Column Chromatography)1. Gravity Chromatography

2. Flash Chromatography

3. High performance Liquid Chromatography

What is column chromatography?• Column chromatography is one of the most useful methods for

purification & separation (Isolation) of individual desire compound from mixture of unwanted compounds.

• It is often used for preparative applications on scales from micrograms up to kilograms

• It is a solid - liquid technique in which the stationary phase is a solid & mobile phase is a liquid.

• The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is Silica Gel, followed by Alumina Oxide.

• The mobile phase or eluent is a liquid. It is either a pure solvent or a mixture of different solvents.

• It can be used for molecules whose molecular weight is < 2000 g/mol

High Performance Liquid Chromatography

Thin Layer ChromatographyThin layer chromatography, or TLC, is a method for analyzing mixtures by separating the compounds in the mixture. (All types of Chromatography works on same principle.)

Uniform layer of Silica Gel coated onto a piece of glass, aluminium or rigid plastic plates are being used for analysis.

The mobile phase moves through the stationary phase and carries the components of the mixture with it.

 Different components travel at different rates.

Application:

To determine the number of components in a mixture,

Identification of compounds, and

Purity of a compound.

Thin Layer ChromatographyIt is used in Analytical study to analyzed microgram (0.000001 g) quantity samples and it takes around 5-10 minutes for result.

Size available:

1. Silica Gel G for TLC (With Binder)

2. Silica Gel H for TLC (Without Binder)

3. Silica Gel GF 254 – with fluorescent indicator

4. Silica Gel HF 254 with fluorescent indicator.

In Thin Layer Chromatography fluorescent is added in stationary phase to analyze substances which are colourless. This we can not see with our naked eyes.

When plate will expose to UV light then it will Glow except the spots of substances & spot will look like a dark patch.

Selection of Stationary Phase & Mobile Phase

• Removal of impurities • No. of components to be

separated • Length of the column

used • Affinity differences

between components • Quality of adsorbent

used

Stationary Phase (Silica Gel or Alumina)

• It requires balancing act between solvent & compound’s polarity.

• The compound must also be soluble in water so they are not permanently adsorbed.

Mobile Phase (Solvents)

• Columns are available in glass tubes, stainless steel & vary in length & diameter.

• Resolution of column depends on both diameter & length of adsorbents packed in column.

• Resolution improves with increase in length & reduces with increase in diameter.

• For e.g. 25 gms of adsorbents will provide a better separation in a 1 cm diameter column than 2 cm diameter column.

• Column dimensions - length & diameter ratio (10:1, 30:1 or 100:1)Column

How Scale Up take place

• Analytical Scale: Column Inner Dia - 4,5,6 mm & it is done at milligram scale.

• Semi Preparative: Column Inner Dia - 10, 20, 30 mm & it is done at milligram scale.

• Preparative: Column Inner Dia – 40, 60, 80, 80 mm & it can be done at gram Scale

• Pilot scale: Kg scale (1, 2, 4, 10, 12 Kg)

• Commercial scale: Bulk Quantity (50, 100, 200, 400 Kgs or more)

Methods of Column PackingDry Method :

Add dry silica / Alumina to the column and apply to the bottom of the column. This will compress the silica gel and keep it compressed for the next steps. Packing can be improved by tapping the column.

While applying vacuum; pour solvent in it.

Allow the solvent to move though the column until reaches to the bottom. At this stage vacuum is not require.

Allow 5–6 columns value of solvent to flow through the column to make sure it is complete packed.

Drain the solvent till the solvent level is just even with the surface of the stationary phase

Methods of Column PackingWet Method:

Fill the column about one third with solvent

In a beaker, measure out the required amount of silica / alumina.

In another beaker, take solvent approximately one and a half times the amount of silica / alumina.

Add silica/alumina to the solvent while swirling in small quantity at a time. Use a glass rod to mix the slurry.

Pour some of the slurry into column & allow solvent to drain to avoid overflowing.

Tap the column carefully to encourage bubbles to rise and the silica to settle

Continue to move the slurry to the column until all the silica or alumina is added.

Wash the inside of the column by pouring solvent down the inside edge.

Drain the solvent till the solvent level is just even with the surface of the stationary phase

How does separation take place?

Types of Columns

Gravity Column Chromatography: Solvent is allowed to move down the column by gravitational forces.

Flash Chromatography:Solvent is pushed down the column by positive air pressure

ApplicationSeparation of mixture of compounds

Purification process

Purification of Phytochemical

Isolation of metabolites i.e. Small molecules

Estimation of drugs

Process DevelopmentPurify Natural compounds

To separate active component from Plant material

Herbal Extraction

Adsorbents used in chromatography method

• Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used for column chromatography.

• These adsorbents are sold in different mesh sizes such as 60-230 mesh, 100-200 mesh, 200-400 mesh & tailor made.

• Adsorbent particle size affects how the solvent runs through the column.

• Smaller particles (higher mesh size i.e. 230-400 mesh) are used for flash chromatography & larger particles (lower mesh size i.e. 60-120/60-200) are used for gravity chromatography.

Difference between Normal Phase & Reverse Phase Chromatography

Normal Phase Chromatography

•  It uses a polar stationary phase and a non-polar (low Polarity Solvents) mobile phase.

•  Non-polar compounds elute faster than polar compounds.

• When we increase polarity of mobile phase elution time will increase.

• It can not be reused / reproducible

• Mobile phase are non polor i.e. IPA, hexane, dichloromethane, chloroform, ethyl ether, and isopropyl alcohol (IPA).

Reverse Phase Chromatography

• It uses a non polar stationary phase and a polar mobile phase.

• Polar compounds elute faster than non polor compounds.

• When we increase polarity of mobile phase elution time will decrease.

• It Can reused / reproducible

• Mobile phase are polor compounds such as water, acetonitrile, methanol

Advantages & Disadvantages of column chromatography

Advantages

It can be used in both analytical and preparative applications.

It is used to identify the number of components of a mixture.

It is also used to separate and purify important quantities of those

components for subsequent evaluation

Any type of mixture can be separated

Any quantity of mixture can be separated

There is wider choice of Mobile Phase (Solvents)

It is low cost process and disposability of the stationary phase once it is used

in the processProcess can be scale up form lab scale

to commercial scale

Automation is possible

Disadvantages

Time consuming Process

More amounts of Mobile Phase (Solvents) required

Scale up process will take a long time to properly prepare & use

Automation makes the techniques more complicated & expensive

Types of Company we need to focus

• Pharmaceutical Industries – Bulk Drugs & API

• Nutraceuticals

• Herbal Extracts products manufacturers

• Research Laboratories

• Laboratories Chemical Repackers

• Contract Research Laboratories

Types of Department we need to contact

• R & D – Research & Development

I. Organic Synthesis Lab,

II. Medicinal Chemistry lab,

III. Novel Drug Discovery,

IV. Clinical Research,

V. Pilot Scale lab,

VI. Preparative Lab,

VII. Semi Preparative Lab

• Q.A. / Q.C.

• Process department / Production Department

• Purchase – At Last

Silent Features Manufacturing since 1973 – Consistence supplies

The product offered is highly active material

Our products has got higher surface area

The product is having better & controlled pore volumes

The Pore diameter is strictly between 50-60A

The bulk density is lower, thus you require less qty of material on column.

The product does not offer hydrolysis of your drugs after separations.

We offer batch to batch reproducible results.

Selectivity & kinetics are maintained constant ( better values)

Higher theoretical plates counts.

Manufactured under strict GMP norms

ISO 9001 accredited manufacturing firm

Comparison Between Silica Gel & Alumina Oxide

Silica Gel Alumina Oxide

Chemical For mula SiO2 (Silicon Dioxide).

Chemical Formula Al2O3 (Aluminium Trioxide ).

it is acidic in nature which we are making it to neutral

It can be Acidic, Basic & Neutral

Silica Gel has Higher Surface area as compare to Alumina i.e. 350-550 m2/gm

Alumina’s Surface Area is less than Silica Gel i.e. 140-160 m2/gm

Sizes available: 35-70, 60-120, 70-230, 100-200, 230-400 mesh

Size Available: 100-300 mesh & 200-400 mesh

Bulk Density: 040-0.65 gm/ml Bulk Density: 0.90-1.2 gm/ml

Pore Dia: 20, 60, 100, 300, 1000A Pore Dia: 50-60A