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mohd's picture

1.can u tel what is the composition of a capillary column(not the stationary phase) hplc colums c18 a single crbon 18 chain is attached to a single sillica molecule.
3.what is the structure of a sillica molecule.(full structure of sillica molecule in a hplc colums). base deactivation is done in hplc columns.(apart from trimethyl silane.
5.for which kind of moleculesgradient compodition is used.(apart from high molecular weight molecules).

Dr. Analytical
Dr. Analytical's picture
Note: This topic was moved

Note: This topic was moved from it's original location.

1. I am not sure which type of capillary column you are asking about.  Do you mean a GC column?  GC columns are made of fused silica, with a polyimide coating.  The fused silica is clear, the polyimide has a brown colr.
2. and 3.  Silica is a three dimensional structure with the chemical composition SiO2.  It is like a cross-linked polymer.  The picture below is from

The surface of silica is covered with SiOH groups, called silanols.  The reaction used to prepare a C18 phase is:

-SiOH + Cl-SI(CH3)2-C18H37   ->   -SiO-Si(CH3)2-C18H37
This reacion is able to react with about 50% of the available silanols.

4. There are many ways to do base deactivation, but usually you can add more carbons to the reagent (the red carbons above) to cover up the silica.
5. Gradient separations are used when the range of retention for your molecules is very large - some elute very quickly while others elute much later, when using a constant mobile phase composition.

mohd's picture

how many molecules of sillica r present in c18 column.and how many chains of c18 r present in a c18 column.

Dr. Analytical
Dr. Analytical's picture
silica is a three-dimensional

silica is a three-dimensional cross-linked polymer, so there is no way to calculate the number of molecules.

The number of C18 chains is difficult to calculate, because it depends on the number of grams of silica in the column, the surface area of the silica, and the reaction effiiciency.  Most chromatographers do not worry about this number.  If you want to compare columns, some manufcturers publish the "% carbon" on the surface.  More carbon usually means more retention.

Lysander's picture
Dear all friends

Dear all friends
Hope all is well with you
Wanted to ask your opinion on this subject
I followed the wash procedures of column wash procedures by Phenomenex catalog for one of their columns;
The column wash procedure was following
95% Water:5% ACN
95%ACN:5% Water
95%ACN:5% Water
95%Water:5% ACN
I did this and at the third phase of THF washing and my back pressure rose to 400 bars-
What could've gone wrong here?
My assumption is that THF could've swelled the end capping of the column-
Now then;
I disassembled the Phenomenex RP C18  column and put a Supelco Supelcosil RP C18 column- And didn't even attempt to use THF for washing-
Here is what happened
I tried 95% Water:5% ACN and my back pressure was running at 150 bars
then I switched to 95%ACN:5% Water and back pressure came down to be about 60 bars- I did this at 0.5 ml/min in reverse flow direction
So far so good!
Then I changed it to 100% ACN and then again my back pressure rose to 350 some bars-
Had I not disconnected it; it would've pop out the plumbing-
Here is what I was running prior to both washes
20mM Potassium Phosphate and 1 ml of Phosphoric Acid in 1 Liter of Water
yielding to gradient mixture with ACN
My questions are the following-
Should I give a memorial service to my two columns?
95% water didn't get rid of the buffer precipitated in the column? I did this for good half an hour
What could've gone wrong here?
Thank you in advance friends-

Dr. Analytical
Dr. Analytical's picture
What is the size of your

What is the size of your columns and what flow rate was used for the washes?  Was it always 0.5 mL/min?  What LC system are you using?  Are there any other mobile phase additives.

Are you certain that you were washing with water and not buffer?  How did you switch from buffer to water?  Was it programmed at the pump (a quaternary system) or did you switch the mobile phase bottles?  (I don't want to sound picky here but these are important details.)

You are correct that the system is behaving like the buffer is still in the column, and is precipitating at high acetonitrile concentrations.  We now need to find out why.
One other thing to try is to repeat each solution without the column attached; pump through tubing only, into a waste container.

Lysander's picture





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Thanks for replying sir
Column sizes were the following
1- 100 mm x 2.6 micron x 4.6 mm and
2- 150mm x 5 micron x 4.6 mm
The flow rates for washes  were 0.5 ml/min-  ( I gradually decreases the flow rates due to back pressure build up- However; it came to the point that my tubings were popping out left and right)
 The analysis was run under 1 ml/min
No- It was not always 0.5 ml/min- Like I mentioned I was brnging it down to prevent high back pressure build up- But it didn't help ( I even came down to 0.1 ml/min)
My LC is lower pressure gradient Dionex ( 400 bar) P680 Pump with ASI 100 Autosampler
PDA 100 Photodiode Array Detector and STS585 Column Thermostat
Other Mobile phase additives? Not as far as I can remember-
20mM Potassium Phosphate and then acidified with 1 ml Phosphoric Acid  in 1 Liter water and Acetonitrile for the organic phase- The  sequence analysis run was performed in Gradient program.
Yes Dr. A
I am 100% sure I was washing with water and not buffer- for switching I just changed 95% Water to 95% ACN - Just like the wash program indicated in the new column booklet-
Dr. A- My bottles are
A- Acetonitrile
C- The buffer I was using which is 20mM Potassium Phsophate +1 ml Phosphoric Acid
( This is the bottle that gets to be changed all the time- The other bottles are only being replenished )
D- Methanol-
I programmed it on   95% B  5% A- I switched to 95%A and 5%B afterwards on steady 0.5 ml/min
On the last note-
I will do as you suggested sir- I will do this tomorrow morning