How Do I Scale Up to a Prep LC Column?

prep column

 

As mentioned in my previous post What is Different about Prep LC?, we ask that you start by developing your method on an analytical scale column. Once you have accomplished this, and wish to start using prep scale HPLC for purification, you will need to determine what size preparative scale (“prep”) column would be most appropriate. Below I have presented a possible scenario for selecting a prep column, based on method development done on an analogous analytical scale column. The following is intended for experienced LC users. Please note that these are my thoughts on how this could be done, based on information that is currently available on the topic. Some laboratories that use prep LC on a regular basis may have other practices that work for their specific application.

 

Determining Factors

Column IDs for our prep columns range from 10 -30 mm, whereas the maximum ID for analytical scale is 4.6 mm. The primary consideration for determining the size of a prep column is the desired capacity for sample loading. Columns have a capacity for sample volume and sample analyte mass, so both should be considered. While some instrument manufacturers address sample load in terms of total sample weight, I will be discussing this in terms of total sample volume, since that translates more directly in practical terms. Theoretical sample volume capacity for our prep columns can be anywhere from 40 µL to about 2 mL, but the practical limit should be determined on a case by case basis for each application.

 

Load Capacity at Analytical Scale

I would start by experimentally determining a practical limit for injection volume on the analytical scale column, with an analytical scale detector in line. For hints on estimating sample injection volumes, please see our FAQs on the subject here: http://www.restek.com/Pages/faq_lc#pkd2. To do this, make a series of injections of a representative sample with volume increasing in increments. (Please see below for an example.) If you are not pleased with results for these injections, try a lower range of volumes. Make sure your representative sample contains the same impurities that you wish to remove from your production scale sample. It will be important also to use a detector that allows you to see both the compound of interest as well as the impurities you wish to remove. Then, come up with a volume that represents an acceptable maximum load volume. Use that number to calculate your scale-up later.

If your prep/production scale sample is expected to be in higher concentrations (in mass of analyte compound per volume units) than you used for the volume test, or perhaps expected to be variable, you should also determine a limit for the mass load at analytical scale. The same technique of doing successive injections in increments will work for this. Examine the peak shape and separation to see what mass of analyte you would be comfortable with loading to get acceptable results for your application. Please keep in mind that practical limits for one analyte may be different from other analytes, based on its unique properties and peak symmetry.

 

Example at Analytical Scale

The analyst has a 150 x 3.0 mm column, and makes successive injections at 2, 5, 10, 20 and 30 µL.

In this case, 20 µL is determined to be the largest acceptable volume, using a sample at concentration of 15 µg/20 µL = 0.75 µg/µL = 750 µg/mL.

Suppose that the sample given to process at prep scale will vary from time to time. The analyst performs some test injections to determine the maximum mass for the compound of interest per injection. For the 150mm x 3.0 mm column, a series of injections is made containing 10, 15, 30 and 60 µg of analyte at the maximum injection volume of 20 µL. Test injections contain 500, 750, 1500 and 3000 µg/mL, respectively.

Suppose, for this example, that a maximum mass load of 3000 µg/mL is established for the injected sample (60 µg per 20 µL). This information will be used in the example following the next section to help determine the size for a prep column.

 

Determining Prep Scale Column Dimensions

The next question to ask is “How much sample do you want to be able to load per injection?” As shown in the FAQ mentioned earlier, optimal injection volumes are directly related to cylinder volume of the column. If you are keeping the same column length, the change in optimal injection volume is proportional to the cross-sectional area, A= πR2, where R is the radius. This would be represented mathematically by this equation:

calculations4

 

 

Please note that some sources suggest overall capacity is equally influenced by column length, but opinions on this vary. For our calculation purposes here, we will assume column length stays the same, so length is not a consideration.

 

Example of Conversion to Prep Scale

Suppose you wish to load a sample volume that is 100 times greater than the maximum volume you determined at analytical scale and your analytical column was 150 mm x 3 mm, then your calculations would look like this:

calculations

 

According to the calculation, you should select a column with radius of 15 mm or larger. A prep column with inner diameter of 30 mm should work for this.

Applying this calculation to the previous example where the maximum volume was 20 µL for a 3 mm ID column, scaling up to a 30 mm ID column would allow you to inject up to 2 mL on the prep column. Suppose that the maximum mass load was 60 µg, or a concentration of 3000 µg/mL for the injected sample. In this case, scaling up to a 30 mm ID column would allow you to inject up to 6000 µg or 6 mg for your compound of interest.

 

Determining Flow Rate

Another significant difference with prep LC is the flow rate. In order to get the best efficiency for a larger ID column, the flow rate must be increased. As was the case for injection volume, the flow rate also is directly proportional to the cross sectional area of the column, so if you’re converting from analytical scale to prep size, you would multiply the initial (analytical scale) flow rate by the ratio of the radius for the prep column squared over the radius of the analytical column squared. (It is equivalent and sometimes easier to use the diameters rather than the radii.) See formula below:

 calculations2

Where F = flow rate, R = radius and D = diameter. A subscript of “1” indicates analytical scale, while subscript “2” indicates prep scale.

As an example, if your flow rate with a 3 mm ID analytical column was 0.4 mL/minute and you are scaling up to a 30 mm ID column, an equivalent flow rate would be calculated this way:

calculations3

If you are not certain whether your LC system can accommodate this increased flow rate, please consult your operating manual or contact the manufacturer to ensure it can handle the desired flow rate.

 

I hope you have found the suggestions in this post useful. Thank you for reading.

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