Ultra-Performance Liquid Chromatography - An Updated Review

 

Kunal Bhattacharya^1,2,3, Nongmaithem Randhoni Chanu⁴*, Atanu Bhattacharjee³,

Bhargab Jyoti Sahariah², Chanam Melody Devi², Ripunjoy Bordoloi²

¹Pratiksha Institute of Pharmaceutical Sciences, Panikhaiti, Guwahati, Assam – 781026.

²NETES Institute of Pharmaceutical Science, Mirza, Kamrup, Assam – 781125.

³Royal School of Pharmacy, The Assam Royal Global University, Guwahati, Assam – 781035.

⁴Faculty of Pharmaceutical Science, Assam Downtown University, Guwahati, Assam – 781026.

 

ABSTRACT:

Ultra-performance liquid chromatography (UPLC) has an advantage over conventional High-performance liquid chromatography (HPLC) as UPLC offers substantial resolution, speed, and sensitivity during analysis. This advanced chromatographic technique uses sub-2μm particles for the stationary phase. As a result, it saves time and reduces solvent consumption, which allows it to take less run time and makes it highly efficient.

 

 

1. INTRODUCTION:

UPLC stands for Ultra Performance Liquid Chromatography^1,2. UPLC is similar to HPLC in principle but one step ahead in speed, sensitivity and resolution^3,4. It uses sub 2μm particle technology⁵ in its stationary phase to reduce the solvent consumption and reduce the overall run time^6,7. As per Van Demeter equation H = A + B/u + Cu, with the help of small particles, peak capacity, and speed during analysis can be improved and taken to new heights, which is the technique used in Ultra Performance Liquid Chromatography. When UPLC is compared to Capillary Electrophoresis and process such as Gas Chromatography decrease in efficiency is observed because low diffusion coefficients are observed in the liquid phase, causing the analytes to diffuse into the stationary phase slowly. The Increased efficiency of UPLC, which can be understood with the help of the Van Demeter Equation, comes with a Disadvantage, which increases backpressure during analysis. To counter this issue, columns with short lengths having sub-2 μm particles are used. To run the separations using columns that are packed with very small particle sizes, UPLC uses the full advantage of chromatographic principles⁸.

 

In recent times, pharmaceutical companies are looking for options that can help them shorten the time and cost required during drug development and analysis by maintaining quality and standards. These are the benefits of rapid analysis done with the help of UPLC. A typical assay can be transferred and optimized using UPLC by attaining both shorter analysis time and increased sensitivity of the analysis.

 

1.1. Principle:

UPLC uses sub 2μm particle technology in its stationary phase⁹. The system follows the principle postulated by Van Demeter equation that basically describe a relationship among the flow rate and height of the plate. The equation for the same is as under:

 

H = A + B/V + CV

Were,

A, B and C are constant

A = Eddy diffusion, H= HETP, B = Longitudinal diffusion, C = Equilibrium mass transfer

V = flow rate.

 

For improvement of the efficiency of UPLC following measures can be taken:

1.     Reduction of the mobile phase's viscosity by applying high temperature, which increases the flow rate significantly, ultimately decreasing the backpressure.

2.     Use of monolithic columns that provides better resolution and increased sensitivity, leading to decreased solvent consumption¹⁰.

 

Chromatographic resolution is described by¹¹:

Rs=√N/4(α-1/) (k/k+1)

 

Were,

Rs = Resolution, α = Selectivity factor, k = Retention factor, N = Separation efficiency

Separation efficiency: (N)

 

N=L/H=L/h dp

Were,

L = Column length, H = Height of therapeutic plate, h = Reduced plate height

dp= Particle diameter

Therefore, Rs α N α 1/dp

 

1.2. Chemistry of small Particles:

Using the sub 2μm particle technology in the stationary phase of UPLC, the efficiency is increased and retained. There is no observation of diminishing efficiency after that. Also, the speed and the peak capacities are taken into new limits with the introduction of smaller particles. As the particle size in the stationary phase decreases, the linear velocity or the flow rate increases. Once the optimized flow rate is obtained, it remains constant. As the particle size in the column increases more than 1.9 μm decrease in efficiency can be seen after a certain period. For proper retention instead of commercially available non-porous small particles, novel porous particles can withstand high pressure. Sol-gel synthesis containing columns with carbon in methyl groups form makes it mechanically strong and efficient and can also work in a wide range of pH¹².

 

2. Instrumentation:

The basic instrumentation of UPLC is given as follows:

 

Fig. 1. Instrumentation and working of Ultra Performance Liquid Chromatography

 

2.1. Pumping Device:

UPLC pumps ideally deliver solvent at around 15000 psi having its highest efficiency across a 15 cm long column that is packed with 1.7μm particles.

Pumps used in UPLC can be classified as¹³:

·       Constant Flow Pump, which is used for all typical UPLC applications.

·       Constant Pressure Pump used mainly for column packing.

 

2.2. Sample Injection:

The introduction of a sample is essential in UPLC. Both manual and automatic injection valves used conventionally do not work correctly under extreme pressure. Therefore, to save the columns from fluctuations in pressure, the injection process should be opted where the process is pulse-free relatively and have a minimum volume of swept to reduce spreading of the bands. In order to capitalize the speed fully achieved in UPLC, there is a requirement of a fast injection cycle time along with a high sample capacity. To increase the sensitivity, the volume of injections should be low, and carryover should be minimal. Upon initiation of injection, to collect the sample from the vial, the flow from the needle is diverted by the inject valve. Thus, the exact volume of the sample is withdrawn from the vial upon which the needle returns to its port. Moreover, there is also a necessary to minimize the sample carryover to the next injection and this is achieved by allowing the washing of the needle for a certain period of time using the washing solution. Besides this, there are also several direct injection techniques that has been mentioned in the literature^14,15.

 

2.3. Detectors:

After separation, the analyte needs to be detected both qualitatively and quantitatively. For this purpose, the UPLC system can be equipped with PDA, ELS, TUV, and FLR detectors individually or in combination¹⁶.

 

2.4. PDA (Photodiode Array) Detector:

This is basically an optical detector that has the capacity of absorbing UV-Visible light ranging from 190-500nm.

 

2.5. ELS (Evaporative Light Scattering) detector:

This technique basically involves the mixing of the solvents with a carrier gas which is inert in its nature. This is followed by forced nebulization after elution is completed which aids in separation of the liquids to minute aerosolized droplets. Moreover, the droplets are also allowed to pass through a heated tube as because the mobile phase evaporates and the droplets becomes smaller that is further pushed by the carrier gas towards the detection zone.

 

2.6. TUV (Tunable Ultraviolet) detector:

The ACQUITY UPLC TUV Detector is a dual wavelength ultraviolet/visible (U.V./Vis) detector and tunable that offers optimal resolution, linearity, and sensitivity separations via UPLC.

 

2.7. FLR (Fluorescent) detector:

This is basically a multi-channel and multi wavelength detector operating in the range of 200-890 nm. Moreover, it also offers a 3D scanning capability for allowing a convenient method development.

 

2.8. Columns:

The packing materials in UPLC columns are approximately 1.7μm which gives the benefit of faster separation. As the bonded stationary phase is required to provide selectivity and retention, the particles are connected in the matrix. Different column types are made by ACQUITY, available in the market, and can be used for the UPLC technique¹⁷.

 

2.9. Commonly used UPLC columns are equipped with the following technologies:

·       BEH technology: BEH C18, BEH C8, BEH phenyl, BEH shielded RP18 columns.

·       Protein separation technology: BEH200, BEH123, BEH450, SEC columns

·       Peptide separation technology: BEH 300, BEH 130

·       Oligoneuclotides separation technology: OST C18 columns.

·       Glycin separation technology: BEH glycan column

·       Charged surface hybrid technology: CSH C18 columns.

 

2.9.1. BEH C18 Columns:

Acquity UPLC BEH C18 columns having a particle size of 1.7um high degree of peak symmetry, chemical stability, and efficiency. They work in the pH range of 1-12 and temperature up to 80^o C¹⁸.

 

2.9.2. BEH C8 Columns:

When compared to C18 columns, C8 columns provide low hydrophobicity, causing lower retention and rapid elution due to the presence of shorter aralkyl chain length. But this column also provides good peak symmetry, stability, and efficiency.

 

2.9.3. BEH shield RP 18 columns:

This variant of columns basically have a polar group that is embedded which in turn combines the hydrophilicity a polar group embedded with the hydrophobicity of a straight- chain alkyl ligand C18.

 

2.9.4. BEH phenyl columns:

Due to pi-pi interactions, complementary selectivity to straight chain alkyl phases is given by this column.

 

2.9.5. BEH200, BEH123, BEH450 Columns:

The determinations are provided accurately by this column which is faster than traditional HPLC SEC assays.

 

2.9.6. BEH 300, BEH 130 Columns:

Because of high-resolution power, these columns' characterization of protein and peptide is improved.

 

2.9.7. OST C18 Columns:

To characterize oligonucleotides by ion-pair reverse-phase chromatography, these columns are used.

 

2.9.8. BEH glycan Columns:

The column of this variant tends to provide an exceptionally high resolutions thereby helping in the improved resolution in characterization of glycoprotein containing compounds.

 

2.9.9. HSS C18 Columns:

These are silica-based C18 columns which helps to give good quality shapes of peak and also, they provide a low pH stability thereby enhancing the retention as compared with the hybrid-based C18 Columns.

 

2.9.10. CSH C18 columns:

These columns are built basically on the platform of charged surface hybrid which offers an enhanced peak resolution at low pH, weak-ionic-strength mobile-phase conditions for most essential compounds¹⁹.

 

3. Applications of UPLC:

3.1. Analysis of trace level priority pesticides in groundwater:

UPLC in association with tandem mass spectrometry (UPLCTM-MS/MS) can be utilized in determination of the content of pesticides present in ground water in a rapid an efficient manner. This combined technique has gained attention in context to its speed during analysis, better resolution and enhanced sensitivity²⁰.

 

3.2. Quantification of bioactive compounds in the tumor‐shrinking decoction:

The bioactive compounds of tumor‐shrinking decoction (FM1523) can be quantitatively determined via UPLC‐MS/MS with MRM mode. Thus, this study could evaluate the pharmacokinetics analysis of TSD during clinical applications and extend a schematic approach for quantifying the bioactive compounds present in the traditional Chinese Medicine decoction²¹.

 

3.3. UPLC in pharmaceutical analysis:

Several essential parameters that are necessary to be evaluated during the manufacturing of a drug products and pharmaceutical dosage form includes the identification, purification, method developments, quantification and also the efficacy and safety. The technique of UPLC is significantly employed in the QA/QC laboratories in quantitative estimation. This is specifically a novel technology allowing newer scope in liquid chromatography with the added benefits of utilizing a minimum runtime and limiting solvent consumption^22-27.

 

3.4. Veterinary drugs quantification in milk:

Several multi-compounds can be analyzed with the help of the most potent measurement tool UPLC–ToF-MS, which can deal with more than a hundred veterinary drugs in the milk. Moreover, during the screening of biological extracts, the use of UPLC is compelling because the additional LC selectivity compensates for the lack of selectivity when compared with the MS/MS option of a QqQ-MS²⁸.

 

3.5. Determination of organic matter in Waste Leachate:

The molecular information for dissolved organic matter in waste leachate can be determined with the help of ultra-performance liquid chromatography, which can be coupled with quadrupole Orbitrap mass spectrometry for better results. Thus, the molecular information for the determination of organic matter could be the fingerprint of storage pits and waste landfills. Furthermore, the data can be further applied in monitoring environments, such as tracing landfill leakage²⁹.

 

4. Advantages of UPLC:

·       UPLC method is more selective, sensitive, and reliable with faster-resolving power and high-resolution performance.

·       The advantage of this method is the justification of drug specificity using LC techniques for various types of active drug compounds. This technique also ensures end-product quality and additionally with a lower operation cost, a reducing processing time and also lowered run time.

·       This technique also provides a rapid analysis using a new column material of remarkably decreased size of particles thereby increasing the sensitivity of the overall analysis.

·       This technique also lowers the consumption of solvent thereby enhancing the throughput of sample. Moreover, it also provides a real time analysis in step with the manufacturing process.

·       It can provide reproducible responses, higher resolution with better shape of peak, and enhanced speed of analysis.

·       Without repacking or regeneration, HPLC columns can be used again to control parameters in a better way without affecting the efficiency of separation.

·       UPLC also supports a lower particle size which in turn provides an enhanced surface area. This combination allows application if high pressure during the solvent flow.

·       UPLC is more practical for worldwide use because of availability of better-quality smaller size porous packing material with the capability of applying a greater pressure.

·       The automation of operating the instrument and also the analysis of data is straightforward.

·       Large-scale procedures can be performed using UPLC^30-35.

 

5. Disadvantages of UPLC:

·       High back pressures compared to conventional HPLC is a significant disadvantage of UPLC, which decreases the lifespan of the columns.

·       Particle sizes of less than 2μm are non-regenerable and can be used for a short time^36,37.

 

6. CONCLUSION:

UPLC provides better resolution and sensitivity and decreases the time of analysis drastically compared to conventional Chromatographic methods. During the Method development of a new drug molecule, UPLC will reduce the time required to optimize the technique. As the procedure takes less time for analysis, it will cause less solvent consumption, making the process economical compared to other methods like HPLC. However, as a disadvantage, high backpressure is seen during the analysis. It can be minimized by increasing the column temperature. Overall, UPLC is a good and promising technique for analysis giving good resolution, speed, and sensitivity.

 

7. DECLARATIONS:

7.1. Competing Interests:

The authors report no conflicts of interest in this work.

 

7.2. Author contributions:

All authors contributed to data collection, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

 

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Accepted on 12.01.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5849-5853.