The Secrets of Rapid HPLC Method Development

The Secrets 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

                        R_s = ¼ N^½ [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

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