Preparative Liquid Chromatography Primer

1. Antioxidant Isolation Using the Prep 150 LC System.
2. Peptide Isolation Using the Prep 150 LC System.
3. Purification of Cannabinoid from Hemp Oil Using the Prep150 LC System.
4. Isolation of Flavonoids from Ginko Biloba Leaf Using the Waters Prep 150 LC System.
5. Isolation of a Natural Product from Echinacea Extract Using the Prep 150 LC.
6. Streamlining Compound Isolation Automatically with UPLC to Prep Chromatography Using Mass-Directed AutoPurification.
7. Mass-Directed Isolation of a Synthetic Peptide Using the ACQUITY QDa Detector.
8. Strategies for Improving Isolation Using Large Volume Leading and Easy Method Development in Prep Chromatography.
9. Techniques for Improving the Efficiency of Large Volume Loading in Prep Liquid Chromatography.
10. Mass-Directed Isolation of Sulfa Compounds from an Antibiotic Mixture with an ACQUITY QDa Detector.
11. Mass Directed Isolation pf a Pharmaceutical Compound Using AutoPurify with an ACQUITY QDa Detector.
12. Typical Conditions for Analyzing and Isolating the Compounds in the Prep Chromatography Mixture Standard with an ACQUITY QDa Detector.
13. Preparatory Chromatography of Natural Product Extracts Utilizing a UV-Based Open Access Walk-up Purification Strategy.
14. A modular Prep HPLC System for the Isolation of Puerarin from Kudzu Root Extracts.
15. Improving Resolution and Column Loading Systematically in Prep Liquid Chromatography for Isolating a Minor Component from Peppermint Extract.
16. Small Scale Peptide and Impurity Isolation Using the ACQUITY UPLC H-Class and Waters Fraction Manager-Analytical Systems.
17. Small Scale Purification of Constituents from Complex Natural Product Extracts Using ACQUITY H-Class and Waters Fraction Manager- Analytical Systems.
18. At-Column-Dilution, Application Notes, Revision A, Waters Corp, 2003.For more information on Useful Application Notes and References, please visit www.waters.com and input the blue reference numbers above in the search box. 

CONCLUSION: 

• At-Column-Dilution – Patented injection technique that increases mass capacity, improves resolution, increases column lifetime, and improves LC system ruggedness by diluting sample in strong solvent at the head of the column. Chromatogram – A graphical or other presentation of detector response or other quantity used as a measure of the concentration of the analyte in the effluent versus effluent volume or time. Column Volume (V_c), V_c = π x (r)² x Height x 66% available to mobile phase / 1000 to convert mm³ to mL – The geometric volume of the part of the tube that contains the packing (internal cross-sectional area of the tube multiplied by the packed bed length, L with compensation for the space occupied by the packing material, 66%), on a small portion of the flow to the detector while the main portion goes to the fraction collector.separations, the dwell volume still exists, but because the mobile phase concentration is constant, there is no observed difference between chromatograms run on different dwell-volume systems. Eluate, Eluent, Elute – Eluate refers to the portion of the eluent that emerges or elutes from the column outlet containing the analytes in solution. In analytical HPLC, the eluate is examined by the detector for the concentration or mass of analytes. In Prep HPLC, the eluate is collected continuously in aliquots at uniform time or volume intervals, or discontinuously only when a detector indicates the presence of a peak of interest. These fractions are subsequently processed to obtain purified compounds.Hydrophobic – A quality possessed by nonpolar radicals or molecules that are more soluble in organic solvents than in water. Ion-exchange chromatography – Ion-exchange chromatography (or ion chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. Separation technique is used for any type of charged molecule such as large proteins, small nucleotides, and amino acids.Isocratic – Mobile phase composition remains constant over the course of a chromatographic separation. Make-up solvent – Solvent used to dilute and transfer sample split from the prep stream to the detectors in a mass-directed purification system. Mobile phase – Solvents used to elute compounds from an HPLC column.Flow rate – Volume of mobile phase passing through the column in unit time. Injector (Autosampler, Sample Manager) – Mechanism for accurately and precisely introducing (injecting) a predetermined volume of a sample solution into the flowing mobile phase stream. Isocratic elution – Composition of the mobile phase remains constant during the elution process. Mobile phase – Fluid that moves, in a definite direction, through the length of the stationary-phase sorbent bed. Peak – Recording of the detector response while a single component is eluted from the column. Resolution (Rs) – Separation of two peaks, expressed as the difference in their corresponding retention times, divided by their average peak width at the baseline. R_s = 1.25 indicates that two peaks of equal width are just separated at the baseline. When R_s = 0.6, the only visual indication of the presence of two peaks on a chromatogram is a small notch near the peak apex. Retention time (RT) – Time between the start of injection or sample introduction and the emergence of the peak maximum. Selectivity – Term used to describe the magnitude of the difference between the relative affinities of a pair of analytes for the specified mobile and stationary phases that comprise the separation system. Scale (Scale-up) – Amount of purified isolate desired per isolation determines the scale, dictates the column diameter, system and hardware requirements. Amount can range from the isolation or purification of small µg quantities of enzymes, to large kg quantities required for industrial drug manufacturing operation. Scale-up is generation of increased quantities of a particular compound for further evaluation following an initial demonstration of potential value. Step gradient – Separation method which maintains the slope before and after a shallow, focused portion of the gradient, thereby preserving the chromatographic profile for these parts of the separation, often used to determine system dwell volume. Silanol – Chemical group bound to the silica substrate column packing material available for interaction with sample. Splitter – Used to divert a small portion of the sample stream to a destructive detector such as in purification with mass spectrometry. Stationary phase – A porous solid (e.g., glass, silica, or alumina) that is packed into a glass or metal tube or that constitutes the walls of an open-tube capillary; mobile phase flows through the packed bed or column, sample to be separated is injected at the beginning of the column and is transported through the system by the mobile phase; different substances distribute themselves according to their relative affinity for the two phases. Throughput – Benefit of operating LC separations at higher linear velocities using smaller-volume, smaller-particle analytical columns, or large-volume, large-particle prep columns; higher resolution, higher speed, and higher efficiency typically deliver higher throughput; larger quantities of compound can be purified per run or per process period.

GLOSSARY

1. System volume – Dwell volume, delay volume, system volume from the point at which the mobile phase solvents are mixed until they reach the head of the column; for high-pressure-mixing LC systems, this comprises the mixer, connecting tubing, and autosampler loop as the primary components, practical importance only for gradient applications.
2. Split ratio – Amount of material continuously “removed” and diluted from the sample stream before transfer to the detectors during mass-directed purification.
3. Size-exclusion chromatography – Chromatographic separation of compounds based on their size in solution.
4. Shallow gradient – Gradient in which the rate of change of organic solvent concentration per column volume is slow, used to separate compounds with similar affinity for the column matrix.
5. Segmented gradient – Separation method that includes several gradients with different slopes to separate multiple compounds with different selectivity with highest throughout.
6. Sensitivity (S) – Signal output per unit concentration or unit mass of a substance in the mobile phase entering the detector, for mass-flow-sensitive detectors, it is the ratio of peak height to unit mass.
7. Reversed-phase Chromatography – Elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.
8. Retention Factor (k) – Measure of the time the sample component resides in the stationary phase relative to the time it resides in the mobile phase; it expresses how much longer a sample component is retarded by the stationary phase than it would take to travel through the column with the velocity of the mobile phase.
9. Prep chromatography – Process of using liquid chromatography to isolate a compound in a quantity and at a purity level sufficient for further experiments or uses.
10. Passive splitter – Device used to continuously sample the main flow or prep stream with a defined split ratio; manages the detector signal in mass-directed purification.
11. Liquid chromatography (LC) – Separation technique in which the mobile phase is a liquid.
12. Inlet – End of the column bed where the mobile phase stream and sample enter.
13. Gradient – Change over time in the relative concentrations of two (or more) miscible solvent components that form a mobile phase of increasing elution strength.
14. Mobile phase modifier – Additives mixed into the chromatographic solvents for improving separation.
15. Mass capacity – Also called “mass load”; maximum amount of material that can be injected onto a column and the resolution of the compound of interest is adequately maintained.
16. Linear gradient – Chromatographic separation method which changes the mobile phase composition at a constant rate over the course of a defined period of time.
17. Ionization – Process by which a molecule acquires a negative or positive charge by gaining or losing electrons to form ions.
18. Hydrophilic – Dissolves or interacts readily with water or polar solvents due to the possession of strong polar groups.
19. Equilibration – For chromatographic methods in which a gradient is employed, post-run equilibration time is necessary to return the column and system to initial mobile phase conditions prior to the next injection. To achieve adequate equilibration, the column should be flushed with 5 times the total column volume (CV), plus 3 times the volume of the system (dwell) at the method starting conditions. Gradient Chromatography Column Re-equilibration, Performance Perspectives, Waters Corp. 1998. Gradient slope – Rate of change in organic solvent composition per column volume during a gradient separation.
20. Efficiency – A measure of a column’s ability to resist the dispersion of a sample band as it passes through the packed bed. An efficient column minimizes band dispersion or band spreading. Higher efficiency is important for effective separation, greater sensitivity, and/or identification of similar components in a complex sample mixture.
21. Dwell volume – system volume from the point at which the mobile phase solvents are mixed until they reach the head of the column; for high-pressure mixing LC systems, this comprises the mixer, connecting tubing, and autosampler loop as the primary components; when mobile phase components are mixed on-line for isocratic.
22. Detector – A device that indicates a change in the composition of the eluent by measuring physical or chemical properties (e.g., UV/visible light absorbance, differential refractive index, fluorescence, or conductivity). Some detectors (e.g., electrochemical, mass spectrometric) are destructive; i.e., they effect a chemical change in the sample components. If a detector is destructive to the sample a splitter is involved to divert.
23. Chromatography – A method of separation in which the components to be separated are distributed between two phases, one of which is stationary (the stationary phase) while the other (the mobile phase) moves relative to the stationary phase.
24. Chromophore – Part of a molecule that absorbs certain wavelengths of visible light and transmits or reflects others. The chromophore is a region in the molecule where the energy difference between two different molecular orbitals falls within the range of the visible spectrum. Visible light that hits the chromophore can thus be absorbed by exciting an electron from its ground state into an excited state.

REFERENCES

• Prep chromatography has become an invaluable technique for pharmaceutical manufacturing, isolation of natural products and other important operations where generation of purified material is required. The key to utilizing the technique to the fullest is directly proportional to the quality of the method development. Once a purification system has been outfitted with the appropriate hardware and software for large scale purification, method transfer from small scale to large scale becomes a matter of routine. 
  • Sarker, S., Latif, Z., Gray, A., “Natural Products Isolation” Second Edition. Humana Press. 2006.
  • Aubin, A. "UV-Directed Purification of a Small-Scale Organic Synthesis," Waters Corporation, Milford, MA USA. 2008.
  • Diehl, D. Fountain, K. Wheat, T. Joblonski, J. Yin, Z. Morrison, D; Collier, S. Murphy, B. Cleary, R. Compagnon, L., "Practical Chemistry Considerations for Purification." Waters Presentation. 2008.
  • Dolan, J. “A Guide to HPLC and LC-MS Buffer Selection.” Advanced Chromatography Technologies. www.ace-HPLC.com.
  • Jablonski, J et.al. “Effective Use of Temperature Control in Compound Isolation.” Waters Corporation, Milford, MA USA.
  • "At-Column-Dilution, Application Notes", Waters Corp, 2003.
  • For more information on Useful Application Notes and References, please visit www.waters.com and input the blue reference numbers above in the search box.