ARTICLE

Case Study in UHPLC Method Transfer

Introduction

The first three articles in our series on HPLC to UHPLC method transfer have outlined the basics of liquid chromatography and method transfer, as well as offering several best practices to ensure optimal data quality and efficiency. In the final article of the series, we will apply these learnings to a real-world analytical scenario to demonstrate the efficiency gains possible when transferring a method from HPLC to UHPLC.

Ginseng, or the root of the panax genus plant, has been utilized in Asia for thousands of years as an antioxidant, anticarcinogenic, anti-inflammatory, antihypertensive and anti-diabetic treatment. The pharmacologically active compounds behind the claims of ginseng’s efficacy are ginsenosides, which are typically broken into two main groups of ginsenosides: the panaxadiol group or Rb1 group that includes Rb1, Rb2, Rc, Rd, Rg3, Rh2, and Rh3; and the panaxatriol group or Rg1 group that includes Rg1, Re, Rf, Rg2 and Rh1.

Qualitative and quantitative analytical techniques for the analysis of ginsenosides are in demand to ensure quality control in ginseng root processing, as well as for the study of their metabolism and bioavailability. Ginsenoside identification and quantification has traditionally been completed utilizing HPLC, but many supplement manufacturers are embracing the efficiency gains associated with UHPLC. In the remainder of this article, we will implement the best practices introduced in prior articles in this series to transfer an HPLC method for the determination of ginsenosides to a UHPLC instrument.

A typical HPLC analysis of ginsenosides utilizes a column with a diameter (dc) of 4.6 mm, a length (L) of 150 mm and particle diameter (dp) of 5 µm, and the following chromatographic conditions:

Table 1: HPLC Method Parameters

ParameterHPLC Value
Expected Analysis Time (tana1)60 minutes
Injection Volume (Vinj1)20 µL
Flow Rate (F1)1.5 mL/min
Isocratic Step Time (tiso1)7 minutes
Gradient Slope (slope1)0.54%/min
Initial Gradient Composition (%Binitial)13%
Final Gradient Composition (%Bfinal)32%

Recalling the information presented in the third article in our series (Best Practices in UHPLC Method Transfer), when converting a method that utilized an HPLC column measuring 150 mm (L) x 4.6 mm (dc) x 5 µm (dp), a 50 mm (L) x 2.1 mm (dc) x 1.9 µm (dp) UHPLC column is typically utilized when transferring the method, as shown in Table 2. A 2.1 mm column diameter is ideal for this analysis, as mobile phase consumption and frictional heating are significantly reduced.

Table 2: Column Dimension Conversions

ParameterHPLC ValueUHPLC Value
Column Diameter (dc)4.6 mm2.1 mm
Column Length (L)150 mm50 mm
Particle Diameter (dp)5 µm1.9 µm

With a comparable column selected, the UHPLC method parameters can now be calculated.  One of the first general parameters that can be calculated is the expected analysis time for the UHPLC run (tana2):

tana2.png

In the ginsenosides example we are evaluating, the HPLC analysis time (tana1) is 60 minutes. The efficiency gains related to the conversion to UHPLC, allowing for a reduction in run time of more than 52 minutes, could yield an additional 55 runs per 8-hour shift (8 runs/shift using HPLC versus 63 runs/shift using UHPLC).

Next, the injection volume of the UHPLC analysis (Vinj2) can be calculated using the column internal diameter (dc) and the length (L):

vinj2.png

The reduced injection volume for the UHPLC analysis (from 20 µL to 1.4 µL) will help to maintain sensitivity and reduce the potential for extra-column band broadening.  This reduced injection volume is also ideal for laboratories and analytical situations where very little sample volume is available.

Once the injection volume has been determined, the mobile phase flow rate (F) is adjusted to maintain a mobile phase linear velocity similar to what was used in the HPLC method.  UHPLC methods typically consume less mobile phase per run, reducing analytical costs and the amount of waste produced in the lab.

f2.png

In the ginsenosides analysis example, the HPLC method consumed 90 mL of mobile phase per run (1.5 mL/minute for 60 minutes), whereas the UHPLC method only consumes 6.2 mL per analytical run, a more than 90% reduction in mobile phase usage.  In addition to the clear cost savings, this reduction in mobile phase consumption can aid users in their environmental and social responsibility goals related to waste reduction.

The calculated mobile phase flow rate for the UHPLC analysis can then be used to calculate the isocratic step time. As noted in the prior article in the series, the ratio between the isocratic step time (tiso) and the column dead time (which depends on the flow rate, column diameter and length) must be maintained between HPLC and UHPLC conditions.

tiso2.png

Next, the gradient slope (slope2) and gradient step time (tgrad2) can be calculated:

slope2.png

tgrad2.png

For the ginsenosides application, the gradient slope would need to be increased 9-fold.

A summary of the new UHPLC method parameters is provided in Table 3.

Table 3: Comparison of Method Parameters Between HPLC and UHPLC Methods in the Analysis of Ginsenosides

ParameterHPLC ValueUHPLC Value
Column Diameter (dc)4.6 mm2.1 mm
Column Length (L)150 mm50 mm
Particle Diameter (dp)5 µm1.9 µm
Expected Analysis Time60 minutes7.6 minutes
Injection Volume (Vinj1)20 µL1.4 µL
Flow Rate (F1)1.5 mL/min0.82 mL/min
Isocratic Step Time (tiso1)7 minutes0.89 minutes
Gradient Slope (slope1)0.54%/min4.29%/min
Gradient Step Time (tgrad) 4.3 minutes

As detailed in this article, the transfer of a method from HPLC to UHPLC can yield significant productivity improvements and cost savings, owing to a reduction in run time and solvent consumption. In many cases, these cost savings and throughput improvements can quickly balance the increased cost of capital associated with UHPLC instrumentation.

For more information on HPLC and UHPLC solutions, please visit: https://www.perkinelmer.com/category/liquid-chromatography-hplc-uhplc-instruments

Source:

  1. Guillarme, D., & Veuthey, P. (2009). UHPLC Instrumentation and Method Transfer Guidelines [Scholarly project]. In PerkinElmer White Paper.