Dr Pramod Kumar Pandey BSc (Hons), MSc, PhD, founder of PharmaGuru.co, is a highly experienced Analytical Research Expert with over 31 years in the pharmaceutical industry. He has played a key role in advancing innovation across leading Indian and global pharmaceutical companies. He can be reached at admin@pharmaguru.co
Learn about column efficiency in HPLC and GC, including its calculation, role in chromatographic methods, and acceptance criteria for optimal system performance
Column Efficiency (Theoretical Plates) In HPLC And GC: A 7 Mintues Easy Learning
Column Efficiency in HPLC and GC tells about the sharpness or narrowness of the chromatographic peak. It is also called the theoretical plate and denoted by N.
Column Efficiency (Theoretical Plates) in HPLC and GC is a key parameter in method development. It plays a crucial role in ensuring the quality and reliability of chromatographic analysis, making it a vital part of system suitability testing. In this article, I will share my in-depth knowledge and practical insights on how you can effectively assess and optimise column efficiency during HPLC and GC analysis.
Through this discussion, you’ll gain a clearer understanding of:
What Column Efficiency (Theoretical Plate) is and why it’s important
How Column Efficiency is calculated for both HPLC and GC systems
The role of Column Efficiency in chromatographic analysis and its impact on results
The acceptance criteria for Column Efficiency and how to interpret them for successful analysis
By the end of this article, you’ll have the tools to apply this essential parameter in your chromatographic methods, ensuring improved performance and precision in your analyses.
Column efficiency or theoretical plate tells about the sharpness or narrowness of the chromatographic peak. It is denoted by N. It is characteristic of the column. It is widely used in system suitability tests in chromatographic analysis, like HPLC and GC.
Figure-1
Source: pharma knowledge forum
As we go from peak-1 to peak-4, peak sharpness decreases, or in other words, peaks are getting broader and broader. Therefore, peak-1 will have a high theoretical plat,e whereas peak-5 will have a lower theoretical plate in the above chromatogram (See figure-1)
Column Efficiency or Theoretical Plate Calculation Formula
There are several formulas available for calculating N. But in industries, the following two formulas are widely used:
Figure – 2
N = 16 (t/w)2
Where t is the retention time and w is the peak width at baseline
or
N = 5.54 (t/w)2
Where t is the retention time and w is the peak width at half height
Formula-1 is used for those peaks which are sharp peaks and symmetrical peaks
Formula-2 is used for broad and peaks unsymmetrical peaks
Case studies for Column efficiency or the Theoretical plate
Non-polar compounds like Naphthalene, Anthracene etc. give sharp peaks in the HPLC and hence for such molecules, formula-1 can be used
Basic compounds or polar compounds like Azithromycin, Erythromycin etc. give broad and unsymmetrical peaks in HPLC and hence in that case formula -2 shroud be used.
Most of the molecules like Methanol, Ethanol, benzene etc give a sharp peak in GC using a capillary column and hence for such molecule formula-1 can be used.
Amino compounds like Aniline, Triethylamine etc. give a broad peak in GC and hence in that case formulae -2 can be used
Acceptance criteria: Column efficiency
Based on the trend data limit should be kept
Generally, for HPLC analysis. N≥2000 for HPLC analysis
But for some molecules like Azithromycin limit of N less than 2000 is also kept with scientific justification. N ≥1500 for Azithromycin.
Column efficiency or Theoretical plate in Chromatographic development
The higher the value of the Column efficiency or the Theoretical plate, better is the method
The higher the value of the Column efficiency or the Theoretical plate, the sharper is the peak
It is one of the SST parameters
The higher the value of the theoretical plate more components can be separated in a single run
Expert Tips: Factors affecting Column efficiency in HPLC
Column efficiency or Theoretical plate depends upon the following factors:
Column Temperature: N isdirectlyproportional to column temperature
Stationary phase purity and chemistry: N depends upon the chemistry and purity of the stationary phase. N is directly proportional to the purity of the stationary phase
Buffer: N is directly proportional to the buffer concentration
pH: Some of the molecules have low N beyond a certain pH range
Peak shape: N is directly proportional to the peak sharpness
Injection volume: N is inversely proportional to the injection volume
Analyte concentration: N is inversely proportional to the Analyte concentration
Peak tailing: N is inversely proportional to the peak tailing
Elution time/Retention time: N is directly proportional to the elution time
Peak width: N is inversely proportional to the peak tailing
Importance of Theoretical plates in HPLC analysis
The concept of theoretical plates is a crucial idea in the context of High-Performance Liquid Chromatography (HPLC). Theoretical plates refer to the number of discrete separation steps (or “plates”) in a chromatographic column, and it is a way of expressing the efficiency of the column in separating compounds.
In an HPLC system, as the mobile phase (the liquid) moves through the column, components of the sample mixture interact with the stationary phase (the material packed inside the column) and get separated based on differences in their affinities for the stationary phase. The more “theoretical plates” a column has, the more efficient it is at separating components, as it essentially means that each “plate” is a site where some of the separation happens.
Let’s break down a theoretical case study to better understand how the concept of theoretical plates is applied in HPLC analysis
ECase Study: Analysing a Sample Mixture containing Two Pharmaceuticals (A & B)
Objective: To determine the separation efficiency of a column when analysing a mixture of two pharmaceuticals: Drug A (a basic compound) and Drug B (a neutral compound), using a reversed-phase HPLC system. The goal is to evaluate the resolution of the two compounds and estimate the number of theoretical plates for the column used in the analysis.
Step 1: HPLC Setup
Column: C18 reversed-phase column, 150 mm length, 4.6 mm internal diameter, 5 µm particle size.
Mobile Phase: A mixture of water (solvent A) and acetonitrile (solvent B) in a 70:30 ratio.
Flow Rate: 1 mL/min.
Detection: UV at 254 nm.
Step 2: Initial Chromatographic Conditions and Results
The chromatogram shows two peaks corresponding to Drug A and Drug B, which are eluted at different retention times (RT). The baseline resolution between the two peaks is calculated as:
Resolution (R)=2x (t2 -t1)/(W1 + W2)
Where:
t1​ = retention time of Drug A
t2​ = retention time of Drug B
W​ = width of the peak at baseline
Step 3: Calculating Theoretical Plates
The theoretical plate number, N, can be calculated using the the following formula -2 of Figure -2:
N = 5.54 (t/w)2
For this case, assume the following:
The retention time of Drug A is 4.5 minutes, and the width of its peak at the baseline is 0.25 minutes.
The retention time of Drug B is 6.2 minutes, and the width of its peak is 0.3 minutes.
For Drug A
NA = 5.54 (4.5/0.25)2 = 1775 theoretical plate
NB = 5.54 (6.2/0.3)2 = 2310 theoretical plates
Step 5: Optimising Conditions
In this case, while the number of theoretical plates is relatively high, there might still be room for optimisation:
Changing the flow rate could influence the efficiency. A slower flow rate might result in better resolution, but it may also increase analysis time.
Adjusting the mobile phase composition (for example, changing the ratio of acetonitrile to water) could improve selectivity and resolution between the drugs.
Column temperature: Increasing the temperature can sometimes improve efficiency by decreasing the viscosity of the mobile phase.
This concept is essential in optimising chromatography conditions and ensuring that separations are performed with high precision and reliability.
Conclusion
I hope this article has helped clarify the concept of column efficiency and theoretical plates in chromatographic analysis, specifically in HPLC and GC method development. Understanding these concepts is essential for optimising separation techniques and ensuring high-quality results in your analytical work. Whether you’re developing new methods or troubleshooting existing ones, knowing how to leverage theoretical plates effectively can greatly improve your separation efficiency and the reliability of your data.
If you have any questions or if there’s something you’d like further clarification on, please feel free to share your thoughts in the comment section below. I’ll make sure to address your queries on a priority basis.
