Hamilton Scale-up Liquid Chromatography
What is Scale-up Chromatography?
Liquid chromatography is a technique used to separate and purify components of a mixture based on their chemical and physical interaction with a stationary phase and a mobile phase. Chromatography is used in many industries like pharmaceutical, biotechnology, food and beverage, environmental analysis, forensics, cosmetics, etc.
Scale-up chromatography is the process or technique of increasing the capacity of a chromatography system to handle larger volumes of sample or to achieve higher throughput. Chromatography is a separation technique used to separate and purify components of a mixture based on their different affinities for a stationary phase and a mobile phase. It is widely used in various industries, including pharmaceuticals, biotechnology, food and beverage, environmental analysis, and more.
Scale-up liquid chromatography involves transitioning from a smaller chromatography system, such as a laboratory-scale system, to a larger system that can handle larger sample volumes, higher flow rates, and higher concentrations of target components. The goal is to maintain the same separation efficiency and resolution while processing larger quantities of sample.
Scale-up Considerations
There are several considerations when scaling up an HPLC method:
- Selecting the appropriate column size
- Choosing the best-suited stationary phase for the application
- Choosing the optimal stationary phase particle size
- Choosing the optimal stationary phase pore size
- Mobile phase selection
- Scaling up and optimizing the mobile phase flow rate
- Ensure the HPLC system is properly designed for the flow, pressure, and dead volumes.
Scale-up chromatography is often performed in the development and manufacturing stages of a product, where larger quantities of purified components are required for further processing or formulation. It is essential to carefully plan and validate the scale-up process to ensure that the desired separation performance, purity, and yield are maintained at the larger scale. Proper scale-up can help streamline production, increase productivity, and ensure consistent product quality.
High Sample Capacity
The sample capacity of a 4.1 x 150 mm PRP-1 column is approximately 10 to 25 mg of DMT protected DNA per run by Ikuta et al.1. When compared to 1 mg/run for a silica C18 column of the same size, the advantage of a polystyrene divinylbenzene becomes clear for higher loading of protected oligodeoxynucleotide purification. Loading capacities up to 500 mg have been obtained on a 21.5 x 250 mm column. A column’s sample capacity is directly proportional to its volume. For example, a 4.1 x 150 mm analytical column has approximately 2.0 mL of volume, while a 21.5 x 250 mm preparative column 90.8 mL. If 10 mg of sample can be purified on the analytical column, the preparative column can purify 45 times that amount or 450 mg.
Column Dimension | Loading Capacity |
| 4.1 x 150 mm | 10 mg |
| 4.1 x 250 mm | 17 mg |
| 10 x 250 mm | 99 mg |
| 21.2 x 250 mm | 446 mg |
| 30 x 250 mm | 892 mg |
| 50 x 250 mm | 2,479 mg |
| 100 x 250 mm | 9,915 mg |
Easy Purification Scale-up
Scaling up from an analytical column to a semiprep or preparative column is easy. The runs below illustrate the reproducible scale-up from a 4.1 mm column to a 100 mm ID column with Cytosine, Uracil, and Uridine.
PRP-1 DNA Analytical Column
4.1 x 250 mm (0.26 mL/min)
PRP-1 DNA Scaleup Column #1
21.2 x 250 mm (7.16 mL/min)
PRP-1 DNA Scaleup Column #2
100 x 250 mm (160 mL/min)
*The flow rate must be increased proportionally to the size of the column when scaling up to larger sizes to maintain analyte retention times of oligomers. The following table is a guideline to determine the appropriate flow rate for a given column hardware dimension.
Flow Rate Scale-up
| Column ID | Flow Rate (mL/min) |
|---|---|
| 1.0 | 0.05 |
| 2.1 | 0.22 |
| 4.1 | 0.80 |
| 4.6 | 1.06 |
| 7.8 | 3.04 |
| 10 | 5.00 |
| 21.2 | 22.50 |
| 30 | 45.00 |
| 50 | 125.00 |
| 100 | 500.00 |
Long Column Life
The poly(styrene-divinylbenzene) stationary phase in PRP-1 and PRP-3 columns is extremely robust and resilient to most HPLC method conditions. This stability prevents column degradation by any of the purification methods in DNA/RNA techniques, assuring reproducible separations and long column life. The inertness of the stationary phase also contributes to the exceptional recovery (>95%) of protected and deprotected oligomers.
Wide Mobile Phase Selection
A wide variety of mobile phases and sample preparation conditions can be used with Hamilton polymer stationary phase columns due to their inertness and full range pH stability.
