Procedure for chromatography involving sample solvent with higher elution strength than the mobile phase.
In preparative chromatography, often the solubility of the sample in the mobile phase is limited, making the mobile phase unsuitable as a solvent for preparation of load. Generally, solvents that have high solubility for the sample also have higher elution strengths than the mobile phase. Additionally, at high loading volumes, these strong sample solvents are known to adversely affect the band profiles leading to poor chromatographic performance. Here, we show that controlling the mobile phase strength during loading and post-load elution resulted in improved band profiles when the sample solvent was stronger than the mobile phase. Such an approach improves performance in preparative chromatography by allowing either higher sample loading or higher organic content in mobile phase (without loss of yield). Alternately, the approach can be used for improvement in performance by increase in yield or product purity.
Patil
Nitin S
NS
Research and Development, Biocon Limited, 20th KM Hosur Road, Electronic City, Bangalore, India. nitin.patel@biocon.com
Mendhe
Rakesh B
RB
Sankar
Ajeet A
AA
Iyer
Harish
H
D000097:Acetonitriles / Q000737:chemistry; D000327:Adsorption; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D000432:Methanol / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide / Q000737:chemistry; D012995:Solubility; D012997:Solvents / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000302:isolation & purification; D014867:Water / Q000737:chemistry
D000097:Acetonitriles; D012997:Solvents; D014867:Water; D012822:Silicon Dioxide; C058028:immunomycin; D016559:Tacrolimus; D000432:Methanol; C032159:acetonitrile