Dr. Pramod Kr. Pandey is a distinguished Analytical Research Expert with over three decades of extensive experience in the pharmaceutical industry. He has contributed his expertise to both leading Indian and global pharmaceutical companies, consistently driving innovation and excellence in analytical research
Learn what resolution in HPLC means, how it’s calculated, key factors affecting it, and its role in method development and system suitability testing
How To Improve Resolution In HPLC: 5 Simple Tips
Understanding Resolution in HPLC: Key Factors and Its Critical Role in Chromatographic Separation
Resolution in HPLC is one of the most important parameters, directly influencing the reliability and accuracy of separation between analytes. It plays a critical role in chromatographic method development and is a key component of system suitability testing (SST), which ensures that the HPLC system is performing within acceptable limits.
In this article, we’ll explore the concept of resolution in detail – how it is defined, how it’s calculated, and why it matters. You’ll also learn about the various factors that influence resolution and the acceptance criteria used to evaluate it. By the end, you’ll have a comprehensive understanding that will allow you to confidently answer questions such as:
What is resolution in HPLC, and how is it calculated?
Which factors affect chromatographic resolution?
Why is resolution critical in HPLC method development?
What are the typical acceptance criteria for resolution in SST?
Resolution is the numerical measurement between two adjacent peaks in the chromatogram. It tells about the separation between the adjacent peaks. It is denoted by R.
In the above Chromatograms, Figure 1, all peaks are well separated, whereas in Figure 2, adjacent peaks are merging. Chromatogram-2 is not acceptable, and hence, resolution-controlling factors must be adjusted to get the separation between the adjacent peaks.
Source: pharma knowledge forumsource: pharma knowledge forum
Resolution Formula
Several formulas are available to calculate the resolution, out of which the following formulas are widely used in pharmaceutical analysis:
Resolution Equation
Where: R is Resolution, w is peak width, N is column efficiency, k is capacity factor, 1 and 2 are peak-1 and peak-2
R-formula-1 is used to calculate the resolution during routine analysis, whereas R-formula-2 is used to modify chromatographic conditions to increase the separation during method development.
Case studies 1:
Generally, good resolution is easily achieved between the non-polar molecules e.g. Naphthalene, Anthracene. In such cases, R more than 2 is easily achieved.
In some typical compound R, less than 1 is also kept. In Erythromycin USP, the Resolution between Related compound n and Erythromycin is not less than 0.8
Case Study 2: Improving Resolution Between Two Closely Eluting Compounds in a Pharmaceutical Assay test
Background: A pharmaceutical lab was developing an HPLC method to analyse two structurally similar active ingredients (Compounds A and B) in a combined tablet formulation. Initial results showed poor resolution (Rs ≈ 0.9) between the two peaks, making quantification unreliable.
Initial Conditions:
Column: C18, 150 mm × 4.6 mm, 5 µm
Mobile Phase: 60:40 Water: Acetonitrile, pH 3.0 (adjusted with phosphoric acid)
Flow Rate: 1.0 mL/min
Detection: UV at 254 nm
Injection Volume: 20 µL
Challenges Identified:
Overlapping peaks with tailing
No baseline separation
Moderate retention time (tR ≈ 4.2 and 4.5 min)
Optimisation Strategy:
1. Changed Mobile Phase pH
pH adjusted from 3.0 to 4.5
Result: Improved ionisation of Compound B → better peak shape
2. Modified Organic Solvent Ratio
Changed to 50:50 Water: Acetonitrile
Result: Increased retention time, sharper peaks
3. Switched Stationary Phase
Moved from C18 to Phenyl column
Rationale: π-π interactions enhanced selectivity between aromatic compounds
4. Reduced Flow Rate
Reduced from 1.0 to 0.8 mL/min
Result: Allowed better interaction time with the stationary phase
Final Conditions (After Optimisation):
Column: Phenyl, 150 mm × 4.6 mm, 5 µm
Mobile Phase: 50:50 Water:Acetonitrile, pH 4.5
Flow Rate: 0.8 mL/min
Injection Volume: 10 µL
Outcome:
Resolution improved to Rs = 2.1
Clear baseline separation achieved
Method passed system suitability criteria for resolution (>2.0)
Successfully validated for routine quality control
Key Takeaways:
Selectivity (α) changes often give the most dramatic improvements when separating similar compounds.
Small adjustments in pH, stationary phase, and solvent ratio can significantly enhance resolution.
Case Study 3: How to calculate resolution in HPLC analysis?
Two analytes were separated using a C18 column. Analyte-1 is eluting at 2.50 minutes, and its peak width is 0.30 minutes. Analyte-2 elutes at 3.10 minutes, and its peak width is 0.35 minutes.
A resolution (Rs) of ≥ 1.5 is generally considered acceptable for baseline separation of two peaks.
Since Rs = 1.85, the peaks are well resolved.
Factors Affecting Resolution in HPLC
The following are the resolution controlling factors in HPLC:
Column chemistry and stationary phase purity: R depends upon column chemistry and the purity of the stationary phase. A column with a pure stationary phase gives high resolution between the peaks
Column length: R is directly proportional to the column length
Particle size: R is inversely proportional to the particle size. On decreasing particle size R increases.
Structure of the molecule: R also depends upon the structure of the molecule. Generally, isomeric molecules have low resolution compared to non-isomeric molecules
Peak shape: R is directly proportional to the peak sharpness
Tailing factor: R is inversely proportional to the tailing factor
pH: R of some of the molecules is pH dependent. Basic molecules have high R at high pH, whereas acidic molecules have high R at low pH
Column temperature: R is the function of column temperature. It may increase or decrease depending on the molecule’s characteristics
Buffer: R is directly proportional to the buffer concentration
Injection volume: R is inversely proportional to the buffer concentration
Sample concentration: R is inversely proportional to the sample concentration
Retention time: R is directly proportional to the retention time
Resolution Acceptance Criteria in HPLC
R should be more than 1.5 or R≥1.5 with baseline separation
In some cases, R< 1.5 should also be acceptable with proper scientific justification
R must be kept based on trend data
How To Improve Resolution In HPLC: 5 Simple Tips
Improving resolution in HPLC (High-Performance Liquid Chromatography) involves optimising several key chromatographic parameters. Here’s a breakdown of the most effective strategies, grouped by the three major contributors to resolution: efficiency (N), selectivity (α), and retention factor (k’), based on the above resolution equation.
Tip-1.Improve Column Efficiency (N)
Use columns with smaller particle sizes (e.g., sub-2 µm for UHPLC).
Increase column length – longer columns provide more theoretical plates.
Minimise system dead volume to avoid band broadening.
Maintain consistent column temperature for reproducibility and sharper peaks.
Use proper injection technique to avoid overloading or band spreading.
Tip-2. Enhance Selectivity (α)
Change the stationary phase (e.g., switch from C18 to phenyl or cyano columns).
Alter mobile phase composition – changing organic modifier (e.g., acetonitrile vs. methanol) or pH can change analyte interactions.
Use ion-pairing agents or buffers to influence the retention of charged compounds.
Adjust mobile phase pH – impacts ionisation state of analytes, especially in reversed-phase HPLC.
Tip-3. Optimise Retention Factor (k’)
Adjust mobile phase strength – lower organic content increases retention (in reversed-phase).
Modify flow rate – slower flow can sometimes enhance separation (though this may affect run time).
Use gradient elution to improve the separation of complex mixtures with wide polarity ranges.
Tip- 4. Stationary phase particle size
On reducing the stationary phase particle size, resolution increases. For example, a C18 column with 3.5 micron particle size will give better resolution than a C18 column with 5 micron particle size
Tip- 5. Other skill-based Tips
Guard column usage to protect the main column and maintain performance.
Proper column flushing and conditioning to avoid contamination or memory effects.
Use temperature control – increasing temperature can reduce viscosity and improve mass transfer.
Applications of Resolution in HPLC Method Development
It tells about the capacity of the method
The higher the R better the method
It is used in SST (system suitability test)
Conclusion
We hope this article has clarified your understanding of resolution in HPLC and the various factors that influence it. With this knowledge, you’ll be better equipped to control these factors during method development, ensuring accurate and reliable chromatographic separation.
If you have any questions or suggestions related to this topic, feel free to share them in the comments section below – we’d love to hear from you!
Resolution is the numerical measurement between two adjacent peaks in the chromatogram. It tells about the separation between the adjacent peaks. It is denoted by R.
How to increase resolution in HPLC?
In HPLC, resolution can be increased by modifying several factors, such as reducing the mobile phase flow rate, increasing the aqueous composition in the mobile phase (in reverse phase mode) and reducing the column stationary phase particle size
References:
Erythromycin USP monograph
Improving Resolution, waters.com
Abbreviations:
R: Resolution
SST: System suitability test
HPLC: High-performance liquid chromatography
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