I'm trying to get conditions for my HPLC experiment!
But, i'm confusing what column is fit for my HPLC condtion.
i want C18 column for analyzing organic compound from my bacteria strain.
i have 4 different columns.
1. SUPELCOSIL™ LC-PAH HPLC Column(5 μm particle size, L × I.D. 25 cm × 4.6 mm) + With guard column
2. SUPELCOSIL™ LC-18-S HPLC Column(5 μm particle size, L × I.D. 25 cm × 4.6 mm)
3. WATERS Atlantis dc18 Column, 100Å, 5 µm, 4.6 mm X 250 mm
4. Hamilton PRP-1 10 µm 100 Å 4.1 x 250 mm
HERE, i have some questions!! please help..
What is differences of SUPELCO PAH & 18-S ? (These two both was subscribed 'C18 (octadecyl) phase' as matrix active group in SIGMA homepage)
What is dC18 in Atlantis column? is that is different from other C18 column?
Hamilton PRP-1 was packed by PSDVB only, is this different from NO C18 polymer ?
Each column, nominated as "C18" or "Octadecyl" is what you ask for.
They differ from each other mainly according to degree of hydrophobicity, stability against harsh eluents and more.
C18 PS-DVB columns are relatively stable to more aggressive, high or low pH eluents /
Silica based columns are to be preferred for using organic solvents.
Sir, this is your research. You have no choice. Study, understand, succeed. I got my PhD this way. There is no compromise.
C18 or ODS is a classification for some columns. Others are amino, C4, C8, C30. But the endcapping is proprietary by the manufacturer and effects hydrophobicity. PSDVB is short for polydivinylbenzene styrene (a polymer) and used for simple sugar analysis.
I suggest you look up the columns in the manufacturer's web site (Supelco, Waters, Hamilton).
PRP-1 is a polymeric reverse phase and runs as reverse phase, in a similar fashion as the C18 columns. Its advantages are that it can run at pH extremes because the there are no silyl ethers to degrade, no silica that dissolves under basic conditions. The Hamilton PRP-1 offering has no C18 bonded to it. It can offer different selectivity as compared to C18 columns. It is run with water/ organic solvent mixtures, and can be run with 100% methanol, acetonitrile, and maybe THF (check the instructions). Other organic solvents may ruin the column because the stationary phase swells.
Silica C18 columns have an octadecyl group bound to the silica. The silica dissolves above pH 7.5, although end-capping reduces the rate of dissolution for a little while. Some columns are end-capped, others are not. The end capping is usually a methyl group; this is done after the reaction with the C18 because the C1 can get between the C18 molecules. There are other end-cappings to make the so-called "AQ" columns which resist phase collapse under highly aqueous conditions. The silyl ethers formed can slowly degrade under acidic conditions, I suggest washing solvent modifiers from the column (such as TFA) after use to extend column life. silica based C18 columns can be used for "non-aqueous reverse phase" for very non-polar compounds. Solvent systems such as methanol/dichloromethane can be used on these columns, which can damage the polymeric reverse phases.
The different C18 columns are made to allow for different selectivities.
Although all your columns are C18 functionalized phases, they will differ in hydrophobicity, peak shape (depending of degree and quality of end-capping and purity of silica) and selectivity and perhaps pore radius. The Hamilton column may give less resolution than the other columns because of the bigger diameter of the spherical particles. Therefore, you will need to test which column is best suited for your analytes.
Hi Joon, Like every other subject from arts, to social sciences , the field of Hplc is also in a phase of continuous growth. There is so enormous literature on the internet, from the basics to quite advanced stages ,That it is fairly with in the reach of every reader. So what I recommend you is to go through the basics of Hplc along with the types of chromatography & the Columns . Due to the expansions in this field, many companies have very commercialized set ups to help you & guide you. The chemistry of the Adsorbents & columns is also expanding quite linearly with time. So pl just spend some time to explore these areas. Dr Abid Aziz Ajaz.
You can use any of the column depending upon the nature and size of your compounds. It will be better first to know the nature of your compounds
HPLC columns have the difference between size, pore and some can withstand more drastic conditions with acid and alkaline solvents. others do not. It depends on the manufacturer and when we choose the columns indicate the manual of them can be used more acidic or alkaline solvents and that pH. I suggest you refer to the manual of each column tag for these specifications. The guard column is a pre column that protects your chromatographic column. The four columns are very good but to Hamilton PRP-1 100 10 micrometres 4.1 x 250 mm has a lower efficiency than can compremeter their separation since this portion is greater, or the peak will be larger.
I suggest using this Supelcosil ™ LC-PAH HPLC Column (5 um particle size, L × ID 25 cm × 4.6 mm) + With guard column but rather look at the catalog to see which the solvent that it supports as you probably the mother should working with buffer.
good job.
I agree with Murad,
it does not necessarily depend on the 'right' column. You can take what you want...you have to do gradient/eluent optimization anyway....In the cases you decribe, there should not be so much difference in your separation. At least, every separation from a crude extract is a compromise...
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