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Preparative HPLC Vs Analytical HPLC: Key Differences Parameter Preparative HPLC Analytical HPLC Purpose To isolate and purify large quantities of a compound Quality control, method development, and content analysis Sample Size Large (milligrams to grams) Small (micrograms to milligrams) Column Size Larger diameter and longer columns (e.g., 10–50 mm ID) Smaller diameter and shorter columns […]
Preparative HPLC Vs Analytical HPLC: Top Interview Questions
What is a prep/preparative HPLC?
Preparative HPLC (Prep HPLC) is a type of high-performance liquid chromatography used to isolate, purify, and collect large quantities of a specific compound from a mixture, typically for further use in research or production.
What is the principle of preparative chromatography?
The principle of preparative chromatography is to separate and purify specific components from a mixture based on differences in their interactions with the stationary and mobile phases, allowing the collection of the target compound in larger quantities.
Which is better, preparative HPLC or analytical HPLC?
Neither is inherently better; analytical HPLC is better for analyzing and quantifying compounds in small amounts, while preparative HPLC is better for purifying and collecting larger quantities of specific compounds. The choice depends on the purpose.
What is Preparative HPLC used for?
Preparative HPLC is used to separate, purify, and collect large quantities of specific compounds from complex mixtures, often for research, pharmaceutical development, or production purposes.
How is Preparative HPLC different from Analytical HPLC?
Preparative HPLC focuses on purifying and collecting compounds in large amounts, using larger columns and higher flow rates. Analytical HPLC, on the other hand, is used for identifying and quantifying small amounts of substances without collecting them.
What types of compounds can be purified using Preparative HPLC?
Preparative HPLC can purify a wide range of compounds, including pharmaceuticals, natural products, peptides, proteins, and synthetic chemicals, depending on their chemical properties.
What are the key factors to consider in Preparative HPLC?
Important factors include column size, flow rate, sample load, solvent selection, and detection method, all of which influence purity, yield, and efficiency of the separation.
Can preparative HPLC be scaled up for production?
Yes, preparative HPLC can be scaled up for pilot or industrial-scale purification, especially in the pharmaceutical industry, though it requires optimization for cost, throughput, and solvent use.
What is the Procedure of preparative HPLC?
Preparative HPLC involves the following 8 steps procedure:
1. Sample Preparation
Dissolve the sample in a suitable solvent (compatible with the mobile phase).
Filter the solution to remove particulates (typically using a 0.45 µm or 0.22 µm filter).
Ensure the sample concentration and volume are appropriate for the column’s loading capacity.
2. System Setup
Choose a preparative HPLC column (larger internal diameter than analytical columns).
Install the column and equilibrate it with the mobile phase.
Set up a collection system (fraction collector or manual collection) for purified compounds.
Flush the column and system with appropriate solvents.
Store the column under recommended conditions.
How do you scaling up from analytical to preparative HPLC?
Scaling up from analytical HPLC to preparative HPLC involves increasing the sample load while maintaining separation efficiency and product purity. It requires careful adjustments to column dimensions, flow rate, injection volume, and method conditions. Here’s a step-by-step approach:
1. Choose the Right Column Size
Scale up the column diameter, not the length.
Use this formula to maintain linear velocity:
2. Maintain Linear Velocity
Linear velocity (flow rate per cross-sectional area) should remain the same as in the analytical method.
Adjust the flow rate according to the new column size to preserve resolution.
3. Adjust Injection Volume
Increase the injection volume proportionally to column volume.
Avoid overloading; perform a loading study to determine the maximum amount you can inject without compromising peak shape or resolution.
4. Use the Same Stationary Phase
Keep the same stationary phase chemistry (e.g., C18, particle size, pore size).
Ensure the preparative column has similar selectivity to the analytical one.
5. Optimise Sample Solvent and Concentration
Sample solvent should be compatible with the mobile phase to prevent peak distortion.
Higher concentrations help reduce injection volume and improve throughput.
6. Use the Fraction Collection System
Set up a fraction collector based on retention times or detector signals.
Collect and pool purified fractions for further use.
7. Validate the Scaled-Up Method
Run a few test injections to verify:
Peak resolution
Recovery
Purity of collected fractions
Adjust flow rate, gradient, or fraction collection windows as needed.
8. Post-Purification Processing
After collection, evaporate solvents or lyophilize to isolate the purified compound.
