Sample load is proportional to the column cross-sectional area. It is necessary to select a column inner diameter suitable for the sample load.Carry out an inspection to verify that the flow rate and the column pressure to be applied at the actual preparative separation are compliance to the system performance. In order to obtain the same separation as the column for investigation, the linear flow rate of preparative column should be same as the column for investigation. Same linear flow rate is obtained by setting the flow rate of preparative column as much as the cross-sectional ratio of the flow rate of the column for investigation. If the linear flow rate is same, the pressure of the preparative column will be the same as the column for investigation. and recovery. A key to perform an efficient preparative separation is to set the elution conditions in consideration of which components should and should not be separated.Packing material with larger particle size compared to analytical purposes compromise the column efficiency to lose required separation. In such case, improve the separation by adjusting elution conditions and/or reduce linear flow rate. If the separation is not improved then, change the column length and/or particle size. Shimadzu strives to improve the purity, speed, and cost efficiency of preparative HPLC.The large-scale preparative system employs an LC-20AP solvent delivery unit. This is a powerful unit offering a maximum flow rate of 150 mL/min that is suitable for automated continuous fractioning with a 20 to 50 mm I.D. column. It can also be used to investigate separation conditions and load conditions and to test the purity of liquid fractions using an analytical column (1 mL/min). Waters offers a wide range of analytical and preparative chromatography columns to meet the needs of virtually every application.The Analytical-2-Prep Column Oven is engineered to manage both analytical and preparative HPLC and SFC columns. Bed dnesities are different in both columns. 

The objective of an analytical HPLC run is the qualitative and quantitative determination of a compound. For a preparative HPLC run it is the isolation and purification of a valuable product. Thus the specific columns are also used in the respective applications.

"Preparative columns" are meant for isolating compounds from natural (product) extracts. It is meant for purifying compounds at a large scale, say in terms of 'milligram' or 'gram'. "Analytical columns" are meant for qualitative analyses. The eluents from the analytical column may not have to be collected. The chromatograms obtained from the use of analytical columns gives qualitative profile or fingerprinting of the sample. Prior to carrying out 'preparative chromatography' runs, it is good to get a profile/fingerprint of the sample by analytical chromatography. Subsequently, the parameters employed in analytical chromatography need to be varied suitable, so as to scale up the process for 'preparative chromatography'. Nevertheless, in some cases, analytical columns can also be used (for preparative-like purposes), to purify compounds, in milligram quantities. Ravi Kant Upadhyay has correctly described the details about the dimensions of the column and other important instrumental features, meant for preparative and analytical chromatography.

It is im portant to say, that very often, the resolution for analytical columns (2-3 mm inner diameter)  is much higher than for prep. Columns (depends of course on the flow rate and the analyte concentration). Thats why I strongly recommend to check the final purity of the compound with analytical HPLC after performing prep. HPLC cleaning.

The elugram can with the same gradient program for both column types is often not 100% identical - for preparative purposes, the flow rate is proportionally higher with respect to the diameter in comparison to analytical column - this simply saves time in the lab! Depends of course on the initial purity of your component. The best opportunity to overcome this issue is to have the same particle diameter in both systems (e.g. 5 µm for analytical and prep. columns - less expensive for analytical column, more expensive for prep. column...).

Phenomenex offers core-shell particle technology (e.g. Kinetex or Kinetex evo) with very good peak resolution even with higher particle sizes. I think this is best for comparable results in analytical and preparative HPLC experiments.

If you are switching from analytical to preparative than it is advise that you should use the same column chemistry in both case no doubt there is a huge difference between columns in each method but you can go with same column chemistry. the difference in sense of column diameter, particle size of packing material, and column loading capacity etc....

I am trying to use an analytic column for purifying small quantity of peptide. However, rerunning the collected fractions does not show me the presence of peptides that I thought I collected. Is there additional steps that I must take? 

Hi Maghesree,

how do you collect the fractions? How much do you inject/how much sample do you have? Please also give information about the instrumentation, e.g. column, solvent system, flow rates etc. Do you freeze-dry the samples afterwards or do any upconcentration step? For our analytical column with 3mm x 250 mm in size, we inject about 0.01 µmol sample, which is nearly "nothing" in terms of workable sample amount (but more than enough for analytical purposes). Are you sure not to overload the column?

Even if you freeze-dry several runs, you will not get more than 1 mg in total, which is hard to recover/aliquot without losing a lot.

You may try semi-preparative columns where you can inject around 10-30 mg per run (depending on the scale of the column).

Marcus

what kind of packing material and packing size used for analytical and for preparative HPLC?

Hi Aiman,

there are several particle sizes (10 µm down to about 2 µm) and architectures (e.g. standard particles, core-shell or monolithic) available, usually the size is smaller for analytical columns and even smaller for UPLC columns. Of course depends on your instrumental setup and what you want to measure.

Best for you would be to check the manufacturer's homepages, they provide more information that I could compress in one post. Search for Agilent, Phenomenex or Waters columns for instance. They also give basic knowledge on HPLC analytics in general, you can learn a lot completely free of charge.

Marcus