Four Critical Aspects of Rapid Method Development and Analysis· Rapid Sample Preparation – minimum steps for maximum effectiveness, use updated tools (combination filters) and multi-sample preparation equipment (SPE 96-well plates)· Choose best bonded phases for high resolution – selecting from typical C18 and C8 bonded phases or those targeted to special sample types·Choose the best column configuration for minimum analysis time with high efficiency and resolution – best column length, internal diameter, p
The Secrets Tips of Rapid HPLC Method Development
Four Critical Aspects of Rapid Method Development and Analysis
· Rapid Sample Preparation – minimum steps for maximum effectiveness, use updated tools (combination filters) and multi-sample preparation equipment (SPE 96-well plates)
· Choose best bonded phases for high resolution – selecting from typical C18 and C8 bonded phases or those targeted to special sample types
· Choose the best column configuration for minimum analysis time with high efficiency and resolution – best column length, internal diameter, particle size
· Using your HPLC instrument to further reduce analysis time and increase sample throughput – optimizing the HPLC and using new features effectively
Bonded Phase Choice Drives Resolution
Changing selectivity (α) influences resolution the most
• Bonded phase is the column choice that controls selectivity
Rs = ¼ N½ [α-1/ α] (k/1+k)
α = selectivity – increase by changing bonded phase and mobile phase
= retention – increase by changing bonded phase and mobile phase
does not improve Rs above k ≈10
Rapid Method Development Scheme – Low pH
Start at low pH for best peak shape, retention and long-term reproducibility
Select starting conditions
· StableBond C18 or C8 for maximum lifetime – Rapid Resolution columns
· pH 1 – 3 with 20 – 50 mM buffer for best peak shape
· Acetonitrile or methanol – start high to scout
· Adjust organic for maximum resolution and retention
· Change organic if resolution not achieved – MeOH or ACN
· Change bonded phase if resolution not achieved – SB-CN, SB-Phenyl, SB-C3
· Use elevated temperature to reduce analysis time further
Rapid Method Development Scouting Chromatograms Change Organic Modifier
· Changing organic modifier can alter selectivity and improve peak shape
· Increase Temperature to Reduce Analysis Time
Rapid Method Development Scheme – Mid pH
· Try pH 7 (pH 6 – 9) with Eclipse XDB-C18 or C8 and follow same process, selecting Eclipse XDB-Phenyl as alternate bonded phase
· Use temperature to reduce analysis time further
Choose Special Bonded Phases for Difficult Samples
· Sample 1 – Highly basic compounds
· May exhibit poor peak shape
· May be difficult to retain
· Column choice 1 – Bonus-RP for better peak shape
· Column choice 2 – Extend-C18 at high pH for better retention and peak shape
Sample 2 – Highly polar compounds
· May be difficult to retain
· May require high aqueous mobile phases
· Column choice 1 – SB-Aq for better retention with high aqueous mobile phases
· Column choice 2 – Bonus-RP for use with high aqueous mobile phases
Sample 1: Highly Basic Compounds
Select Bonus-RP for Improved Peak Shape
Extend-C18 Improves Retention and Efficiency of Procainamides at High pH
Sample 2 – Highly polar compounds
Select New ZORBAX SB-Aq Columns for Method Development in High Aqueous Mobile Phases
Good retention for polar compounds in high aqueous mobile phases
· Reproducible retention without “phase collapse”
· Different selectivity vs. conventional C18 columns
· Highly, stable at low pH and high temperature (up to 80°C)
Rapid Resolution Columns Reduce Method Development and Analysis Time
· Rapid Resolution columns (3.5 µm) are available in many configurations
· Available in analytical, narrow bore, microbore, and capillary internal diameters for compatibility with any sample size.
· Semi-preparative and preparative columns use 5 µm particles.
· Rapid Resolution columns reduce isocratic and gradient run times with:
· Shorter column lengths
· Higher flow rates
· Optimized HPLC instrument
Rapid Resolution Columns Reduce Isocratic Analysis Time by 50% or More
Higher Flow Rates Reduce Analysis Time with Rapid Resolution Columns
Gradient Analysis Time = Run Time + Equilibration Time (single step return)
Short, Rapid Resolution Columns Reduce Gradient Analysis Time
Gradient Time α Column Length
Summary
· C18 and C8 bonded phases are the best for initial rapid method development with typical sample types
· Choosing the most sample appropriate bonded phase and using special, targeted bonded phases, such as SB-Aq for polar, difficult
to retain compounds can decrease method development time
· Rapid Resolution columns are needed to reduce method development time
· Rapid Resolution columns reduce both gradient and isocratic analysis time and permit high throughput rapid analysis
· Rapid Resolution columns can work effectively with your HPLC
· HPLC instruments may have additional capabilities to speed up method development and reduce analysis time
Reference: Agilent