Column ChromatographyColumn Chromatography

Types of columns:Types of columns:

1- Gravity Columns:1- Gravity Columns:The mobile phase move through the stationary phase by The mobile phase move through the stationary phase by gravity force.gravity force.

2- Flash Columns (Air or nitrogen pressure):2- Flash Columns (Air or nitrogen pressure):The mobile phase is pushed by stream of air or nitrogen The mobile phase is pushed by stream of air or nitrogen using special using special values (Adaptors).using special using special values (Adaptors).

                                                                                                                                                                                                                                                    

            

3-Low and Medium Pressure Columns 3-Low and Medium Pressure Columns (pumped):(pumped):The movement of mobile phase is accelerated by using The movement of mobile phase is accelerated by using pumps that generate low or medium pressure. The pumps that generate low or medium pressure. The increase in the flow rate shorten the time of separation.increase in the flow rate shorten the time of separation.

4-Vacuum Columns [Vacuum liquid chromatography 4-Vacuum Columns [Vacuum liquid chromatography (VLC)]:(VLC)]:

The adsorbent is applied dry into a sintered glass funnel. The adsorbent is applied dry into a sintered glass funnel. The sample is applied by dry method or as solution. The sample is applied by dry method or as solution. Then the mobile phase is added portion by portion and Then the mobile phase is added portion by portion and vacuum is applied after each portion to collect each vacuum is applied after each portion to collect each fraction.fraction.

5- High pressure Columns (HPLC):5- High pressure Columns (HPLC):In this columns we use very fine silica gel so In this columns we use very fine silica gel so great increaser in separation power. great increaser in separation power. However, the flow rate of the mobile phase is However, the flow rate of the mobile phase is severely decreased. High pressure pumps are severely decreased. High pressure pumps are used to push the solvent through the column used to push the solvent through the column which in this case must be made of stainless which in this case must be made of stainless steel.steel.

Backing of Columns:Backing of Columns:The adsorbent is applied to the Column in two The adsorbent is applied to the Column in two

ways:ways: Slurry packing (Wet method): Slurry packing (Wet method):

The adsorbent is suspended in the mobile The adsorbent is suspended in the mobile phase and stirred very well to drive off all air phase and stirred very well to drive off all air bubbles. The resulted slurry is then poured bubbles. The resulted slurry is then poured into the column. At the tap end of the into the column. At the tap end of the column a piece of glass wool or cotton must column a piece of glass wool or cotton must be added before the slurry application. Sand be added before the slurry application. Sand may be added after the slurry. After slurry may be added after the slurry. After slurry application the column must be allowed to application the column must be allowed to settle overnight.settle overnight.

In gel chromatography the adsorbent must In gel chromatography the adsorbent must be soaked in the mobile phase overnight to be soaked in the mobile phase overnight to absorb the mobile phase and swell.absorb the mobile phase and swell.

2- Dry Packing: 2- Dry Packing:

In this method the dry adsorbent is poured In this method the dry adsorbent is poured to the column directly. Vibration is the to the column directly. Vibration is the applied to get rid of air bubbles then the applied to get rid of air bubbles then the mobile phase as passed through the mobile phase as passed through the adsorbent. This method can not be applied adsorbent. This method can not be applied gel Chromatography.gel Chromatography.

Mobile phase:Mobile phase:It is a mixture of organic solvents (unusually It is a mixture of organic solvents (unusually one solvent only) the choice of the column one solvent only) the choice of the column mobile phase is achieved after TLC study in mobile phase is achieved after TLC study in different solvent systems. Good solvent different solvent systems. Good solvent system must produce system must produce RRf f value less than 0.6 value less than 0.6 for all materials to be separated by the for all materials to be separated by the column. If the system moves them more and column. If the system moves them more and produces higher produces higher RRff no separation will occur. no separation will occur. Systems that do not move spots at all on Systems that do not move spots at all on TLC are not good for column separation. TLC are not good for column separation.

Isocratic system:Isocratic system:

Means using the same mobile phase from the Means using the same mobile phase from the beginning to the end of the separation.beginning to the end of the separation.

Gradient:Gradient:

The polarity of the system increased gradually The polarity of the system increased gradually during separation by increasing the during separation by increasing the proportion of the more polar solvent. A typical proportion of the more polar solvent. A typical gradient may be start with CHClgradient may be start with CHCl33, followed by , followed by CHClCHCl33/MeOH mixtures with gradual increase in /MeOH mixtures with gradual increase in % of MeOH till all spots are eluted from the % of MeOH till all spots are eluted from the system.system.

Monitoring the column:Monitoring the column:Usually fractions of certain volume are Usually fractions of certain volume are

collected evaporated to small volume collected evaporated to small volume and spotted on TLC. Similar fractions and spotted on TLC. Similar fractions are collected together for more are collected together for more purification or crystallization.purification or crystallization.

In bioassay guided fraction the fractions In bioassay guided fraction the fractions are monitored by the bioassay then by are monitored by the bioassay then by TLC.TLC.

Monitoring the column:Monitoring the column:

Usually fractions of certain volume are Usually fractions of certain volume are collected evaporated to small volume and collected evaporated to small volume and spotted on TLC. Similar fractions are spotted on TLC. Similar fractions are collected together for more purification or collected together for more purification or crystallization.crystallization.

In bioassay guided fraction the fractions are In bioassay guided fraction the fractions are monitored by the bioassay then by TLC.monitored by the bioassay then by TLC.

Sample Application Sample Application 1- Wet application: Dissolve the sample in 1- Wet application: Dissolve the sample in

the initial mobile phase and apply by the initial mobile phase and apply by pipette to the top of the column. This is pipette to the top of the column. This is very good method but in most of cases the very good method but in most of cases the samples are not soluble in the initial samples are not soluble in the initial mobile phase.mobile phase.

2- Dry loading: Dissolve sample in any 2- Dry loading: Dissolve sample in any

volatile solvent. The sample solution is volatile solvent. The sample solution is then adsorbed on small weight of then adsorbed on small weight of adsorbent and the solvent is allowed to adsorbent and the solvent is allowed to evaporate. The dry adsorbent loaded with evaporate. The dry adsorbent loaded with the sample is then applied to the column. the sample is then applied to the column.

Theoretical cencoptsTheoretical cencopts 1- 1- Differential migration:Differential migration:

Different compounds move through the Different compounds move through the system with different rates of system with different rates of movement this is called "differential movement this is called "differential migration". The speed of any compound migration". The speed of any compound in the mixture is determined by the in the mixture is determined by the number of molecules of that compound number of molecules of that compound in the mobile phase. in the mobile phase.

Suppose we have mixture of materials “A” and Suppose we have mixture of materials “A” and “B”:“B”:

A: Have more affinity to mobile phase ei large number A: Have more affinity to mobile phase ei large number of molecules are present in the mobile phase.of molecules are present in the mobile phase.

B: Have more affinity to stationary phase ei small B: Have more affinity to stationary phase ei small number of molecules are present in the mobile phase.number of molecules are present in the mobile phase.

Stationary Phase

Mobile Phase

A B

B

A

Bs

Bm

As

Am

Mathematical prsentation of Mathematical prsentation of differential migration:differential migration:

U: velocity of solvent (mobile U: velocity of solvent (mobile phase).phase).

Ux: velocity of material X.Ux: velocity of material X.

R: fraction of material X in the R: fraction of material X in the mobile phase.mobile phase.

Ux = URUx = UR

If R = I ie all the compound molecules are If R = I ie all the compound molecules are present in the mobile phase. present in the mobile phase.

Ux = U X IUx = U X I

Ux = uUx = u

:. Matenial X will move with the solvent velocity. :. Matenial X will move with the solvent velocity.

if R = 0.0 ie all the compound molecules are if R = 0.0 ie all the compound molecules are present in the stationary phase. present in the stationary phase.

Ux = u x 0.0Ux = u x 0.0

:. Ux = 0.0:. Ux = 0.0

:.Material X will not move at all. For any material to :.Material X will not move at all. For any material to be separated through the system it must be be separated through the system it must be distributed between the mobile phase and distributed between the mobile phase and

stationary phase.stationary phase.

Capacity Factor:Capacity Factor: The factor that control the distribution of The factor that control the distribution of

any material between the two competitive any material between the two competitive phases is called “Distribution coefficient” or phases is called “Distribution coefficient” or “Capacity factor” or “Mass distribution “Capacity factor” or “Mass distribution ratio” K’ ratio” K’

(n)s(n)s

K’ = K’ =

(n)m(n)m(n)s : Is the total number of moles of the compound in (n)s : Is the total number of moles of the compound in the stationary phase.the stationary phase.

(n)m : Is the total number of moles of the compound (n)m : Is the total number of moles of the compound in the mobile phase.in the mobile phase.

ttrr – t – t00

K’ = K’ =

tt00

ttrr : Time required for the sample to : Time required for the sample to cross the column (retention time). cross the column (retention time).

tt00 : Time required for the solvent : Time required for the solvent molecules to cross the column. molecules to cross the column.

Bigger K’ means that the material Bigger K’ means that the material

retained more time on the column ei retained more time on the column ei move slowly. Smaller K’ means faster move slowly. Smaller K’ means faster movement.movement.

2- Movement of materials through the 2- Movement of materials through the chromatographic system in the form of “zones” chromatographic system in the form of “zones”

or “bands”:or “bands”: It was assumed that the chromatographic system It was assumed that the chromatographic system

composed of number of “distribution systems” or composed of number of “distribution systems” or “equilibrations” called “Theoretical Plates”. Each “equilibrations” called “Theoretical Plates”. Each theoretical plat is composed of stationary phase and theoretical plat is composed of stationary phase and mobile phase. The height of each plate is called mobile phase. The height of each plate is called “Height equivalent to Theoretical Plate” (HETP).“Height equivalent to Theoretical Plate” (HETP).

The number of theoretical plates “N” is important for The number of theoretical plates “N” is important for separation. Increasing “N” resulted in narrower bands separation. Increasing “N” resulted in narrower bands

and better separation.and better separation.

LL

NN = =

HETPHETP

L: Column lengthL: Column length

““N” can be increased by: N” can be increased by:

1- Increase the length of the column 1- Increase the length of the column (impractical).(impractical).

2- Decrease the HETP.2- Decrease the HETP.

How to decrease HETP: How to decrease HETP: Decrease the particle size of the stationary phase. Decrease the particle size of the stationary phase. Proper selection of good mobile phase.Proper selection of good mobile phase.

Materials move through the column as bands or Materials move through the column as bands or zones and velocity is controlled by K'.zones and velocity is controlled by K'.

Material with K' = 1 (64 molecules)Material with K' = 1 (64 molecules)

The material will be present in the middle of the The material will be present in the middle of the system in the form of band. If we increase “N” system in the form of band. If we increase “N” the material will from narrower. Narrow bands the material will from narrower. Narrow bands allow better separation of mixtures.allow better separation of mixtures.

32 32

2 2

16 16

16 16

16 16

12 12

12 12

12 12

8 8

8 8

8 8

8 8

4 4

4 4

2 2

2 2

12 12

8 8

8 8

2 2

N= 5 N= 25 N= 150

N= 5 N= 25 N= 150

A

BB

A A

B

Factors affecting Factors affecting separation:separation:

Factors due to Stationary Phase:Factors due to Stationary Phase:

1- 1- Particle size of the stationary phase: Reducing Particle size of the stationary phase: Reducing the particle size increases the surface area the particle size increases the surface area and improve separation. However, reduction and improve separation. However, reduction of the particle size will decrease the flow rate of the particle size will decrease the flow rate of the mobile phase.of the mobile phase.

In HPLC we use very fine particles to get very In HPLC we use very fine particles to get very good separation. The flow rate problem is good separation. The flow rate problem is solved by the use high pressure pumps to solved by the use high pressure pumps to push the mobile phase through the stationary push the mobile phase through the stationary phase. Columns are made of stainless steel phase. Columns are made of stainless steel to withstand the high pressure. to withstand the high pressure.

2- 2- Adsorbent activity: The choice of the Adsorbent activity: The choice of the suitable adsorbent is very important.suitable adsorbent is very important.

3- Uniformity if packing of the column: If the 3- Uniformity if packing of the column: If the stationary phase is not packed uniformly stationary phase is not packed uniformly then the bands will be irregular and less then the bands will be irregular and less uniform resulting in poor separation.uniform resulting in poor separation.

4- Concentration of the mixture: the proper 4- Concentration of the mixture: the proper ratio between sample to be separated and ratio between sample to be separated and the amount of stationary phase is very the amount of stationary phase is very important too much samples resulted in important too much samples resulted in bad separation.bad separation.

Factors due to Mobile Phase:Factors due to Mobile Phase:

1- 1- Selection of the proper mobile phase: Very polar Selection of the proper mobile phase: Very polar mobile phase will wash out all components without mobile phase will wash out all components without any separation. On the other hand very non polar any separation. On the other hand very non polar mobile phase will result in broad band and poor mobile phase will result in broad band and poor separation.separation.

2- Rate of flow: Slower flow rate usually resulted in a 2- Rate of flow: Slower flow rate usually resulted in a better separation and narrower bands.better separation and narrower bands.

3- Consistency of flow: The continuous flow of the mobile 3- Consistency of flow: The continuous flow of the mobile phase during the whole experiment gives better phase during the whole experiment gives better separation than interrupting the flow then continue it separation than interrupting the flow then continue it later.later.

Factors due to Columns:Factors due to Columns:

Column dimensions: Increasing the length Column dimensions: Increasing the length of the column improve separation. of the column improve separation. However, that usually leads to slower flow However, that usually leads to slower flow rate. Also increasing the column length rate. Also increasing the column length some times is impractical.some times is impractical.

Column temperature: Increasing the Column temperature: Increasing the temperature usually reduces the adsorption temperature usually reduces the adsorption power of the stationary phase and increase power of the stationary phase and increase elution speed. This may leads to decrease elution speed. This may leads to decrease in the efficiency separation.in the efficiency separation.