INTRODUCTION:

High Performance Liquid Chromatography (HPLC) is the widely used liquid chromatographic technique in the qualitative and quantitative analysis of drugs. It is also used for the identification and quantification of compounds in the process of drug development and has been used over the world since decades. The principle of separation of compounds is given by Van Deemter equation, which explains the relationship between linear velocity (flow rate) and plate height (HETP, column efficiency).

According to this, as the particle size of column material decreases, the efficiency of the separation, speed and resolution also increases. However certain analytical needs cannot be fulfilled by HPLC method such as determination of complex samples such as biological samples, degradation products, impurities, formulation excipients, drug metabolites and drug isomers. In HPLC method problems arises related to determination of analytes at low concentration (0.1%), speed of analysis and resolution per unit time. To achieve improvement in resolution, speed and sensitivity in LC, a new system design with significant advancement in the instrumentation and column technology has been developed based upon sub 2micron particles called Ultra High Performance Liquid Chromatography (UHPLC) which, is also known as UPLC (Ultra Performance Liquid Chromatography). UPLC is a new category of analytical separation science that retains the practicality and principles of HPLC, while increasing the attributes of speed, sensitivity and resolution.^1,2

Now-a-days pharmaceutical industries are focusing for new ways to increase economy and shorten time for drug development. The separation and quantification in UPLC is done under very high pressure (up to 100M Pa). When compared to HPLC, under high pressure no negative influence is observed on analytical column. And also other components like time and solvent consumption is less in UPLC. In the year 1999, Waters developed the Hybrid Particle Technology (HPT) column for HPLC, which has high mechanical strength, efficiency, pH stability and peak shape for basic compounds. The second generation hybrid material particle composed with Bridged Ethyl siloxane/silica Hybrid (BEH) structure was developed which provides improved efficiency, strength and pH range. High strength silica (HSS) particle technology has also been used which provides increased retention time and selectivity of compounds compared to hybrid particles. The latest advancement in hybrid materials was Charge Surface Hybrid (CSH) Technology which contains surface charge within the packing materials to provide enhanced selectivity and better peak shape. From the above development in column packing material and particle size, Waters Company was given the trade name of UPLC.^{1, 2}

PRINCIPLE:

The principle of separation in HPLC and UPLC is based on the van Deemter relationship. It explains the correlation between flow rate and plate height. The equation is

H=A + B/ u + C u

Where H = Height Equivalent to Theoretical Plate (HETP); A = Eddy diffusion; B = longitudinal or axial diffusion; C = Solute’s mass transfer; u = flow rate (linear velocity) of the mobile phase

The A term is related to the particle size of the packing material. Its value is determined by how well the chromatographic bed is packed. Also it is related to the uniformity or non-uniformity of the flow to and around a particle. The A term is independent of velocity. It is smallest when the packed column particles are small and uniform. The B term represents axial diffusion or the natural diffusion tendency of analyte in the bulk mobile phase and on the stationary phase. It decreases with increasing flow rate of mobile phase [linear velocity]. This effect is diminished at high flow rates and so this term is divided by u. The C term is related to both the linear velocity of the mobile phase and the square of the particle size. It is the interaction of analyte molecules with the internal surface of the stationary phase and their distance of diffusion into and out of the pores of the packing material.^1,
2

Factors influencing separation:

· Parameters affecting efficiency:

· Column length

· Particle diameter

· Particle size distribution

· Flow rate

· Parameters affecting retention factor:

· Eluent type

· Eluent composition

· Stationary phase type

· Analyte nature

· Parameters affecting selectivity:

· Stationary phase type

· Analyte nature

· Eluent additives

· Temperature

· Eluent composition (ionizable analytes)

The quality of the column packing, the particle size and the column dimensions play a part in determining the column efficiency. Longer columns gives narrower peaks (more efficient) but analysis time also increases significantly so this should be intuitive. Smaller particles generally provide more surface area and better separations giving narrower peaks.³ Temperature decreases viscosity of the solvent and reduces back pressure and also diminishes retention. For a variation in temperature by 10°C, the retention time decreases by about 20% in isocratic chromatography. Efficiency increases (peak shape improves) as flow rate decreases but baseline resolution between the peaks of interest cannot be achieved. Stationary phase properties like hydrophobicity, polarity and nature of the base silica play a key role in the physicochemical interaction with the analyte thereby affecting the selectivity of a separation.⁴

Advantages of HPLC:

Sensitivity and accurate quantitative analysis.⁴

· Able to separate non-volatile and thermally unstable compounds.⁵

· HPLC has the ability to separate, and identify compounds that are present in any sample that can be dissolved in a liquid in trace concentrations as low as parts per trillion.

Disadvantages of HPLC:

· Co-elution, adsorbed compounds, cost and complexity.⁶

Advantages of UPLC:

· Decrease in consumption of mobile phase volume by at least 80% compared to HPLC.

· Decreased run time and cost of operation.^7,
8

· Lower injection volume is required.⁹

· Greater Signal to Noise ratio (S/N) due to the reduction in band broadening thereby increasing the sensitivity.

· Because of better chromatographic peak resolution, the problem of ion suppression from co-eluting peaks is greatly reduced.

· Faster resolving power.¹⁰

· The higher column temperature minimizes the mobile phase viscosity resulting in the high diffusion coefficient without significant loss in efficiency and increase in column back pressure.

Disadvantages of UPLC:

· Higher back pressures compared to conventional HPLC which decreases the life of the columns.

· The particles of less than 2 μm are mostly non-regenerable therefore, have a narrow use.¹¹

Analytical method validation:

Validation should demonstrate that the analytical procedure is suitable for its intended purpose. The following validation characteristics were addressed:

· Specificity

· Accuracy

· Precision (repeatability, intermediate precision, and reproducibility)

· Detection Limit/Limit of detection (LOD)

· Quantitation Limit/Limit of quantification (LOQ)

· Linearity

· Range

· Robustness

System Suitability Tests: The system suitability test was used to ensure that the chromatographic system and procedures are adequate for the analysis performed. Parameters of this test were retention time, column efficiency (number of theoretical plates), asymmetry of chromatographic peak (tailing), resolution and reproducibility (in terms of Relative Standard Deviation of peak area)¹²

Applications of HPLC:

· Drug assay

· Stability testing

· Chiral separations

· High-throughput screening

· Analysis of pollutants and food‐relevant compounds

· Bioanalytical separations such as in proteomics^{6, 13}

· Steroid analysis¹⁴

· Forced degradation studies and impurity testing¹⁵

Applications of UPLC:

· Raw material, inprocess and finished product Quality Control (QC)

· Method development and validation

· Forced Degradation Studies (FDS)

· Dissolution Testing and

· Bioequivalence/Bioanalysis Studies

· Toxicity Studies

· Therapeutic Drug Monitoring

· Analysis of Contaminants in Foodstuffs

· Peptide mapping¹⁶

· Analysis of pesticides in groundwater¹⁷

· Detection of Metabolites¹⁸

Comparison between HPLC and UPLC:

· Van Deemter plot comparing particle size

UPLC column with 1.7 µm particles provide 2-3´ lower HETP (H) values compared to HPLC columns with 3.5 µm particles. Additionally, these lower H values are achieved at a higher linear velocity and over a wider range of velocities. This means that mass transfer is improved dramatically with the smaller particle, resulting in better efficiency and resolution. It also means that an increased range of linear velocities can be used to gain this improved performance. Separations are able to be performed at faster linear velocities, thus increasing speed of analysis, without compromising resolution.

Figure 1 Van Deemter plot comparing particle sizes used in HPLC and UPLC¹⁹

Comparison between HPLC and UPLC considering instrument related parameters:

Table 1 Table showing differences between HPLC and UPLC

Characteristics	HPLC	UPLC
Particle size	3 to 5µm	Less than 2µm (1.7μm)
Maximum backpressure	300-400 bars	1000 bars
Analytical column	C18	UPLC BEH C18
Column dimensions	150 X 3.2 mm	50 X 2.1 mm
Injection volume	5µL	2µL
Column temperature	30 ° C	65 °C
Total run time	10 minutes	1.5 minutes
USP resolution	3.2	3.4
Plate count	2000	7500
Flow rate	3.0 ml/min	0.6ml/min
Pressure	Up to 6000 psi	Up to 20000 psi

Band spreading effect on HPLC compared to UPLC:

· Effect on peak shape: Reducing band spreading results in improvements in efficiency and sensitivity

Figure 2 Figure showing effect of band spreading on peak shape in HPLC and UPLC columns¹⁹

· Effect on column performance: When extra-column band spreading is minimized as in the UPLC Instrument, the theoretical performance of a column can be achieved.

Figure 3 Figure showing impact of instrument band spreading on column performance.

(ACQUITY UPLC BEH C18 2.1 x 50 mm, 1.7 µm column; flow rate = 0.4 mL/min) ¹⁹

· Run time comparison between HPLC and UPLC: Short column lengths (30-50 mm) used in UPLC will give short run times compared to HPLC columns (100 - 250mm).

Figure 4 Figure showing difference in run time between chromatograms of melatonin in rice extracts obtained by HPLC and UPLC²⁰

In this study four caffeine metabolites are analyzed using the same chromatographic conditions [except for flow rate as noted] on a fully-optimized, microbore HPLC instrument vs. a standard ACQUITY UPLC Instrument. The improvements in efficiency, resolution, peak shape and height illustrate the benefits of UPLC Technology.¹⁹

Flexibility in improving resolving power in peptide mapping: Peptide mapping is an essential technique for the characterization of proteins. Due to exceptionally reduced instrument and column dispersion, the analysis of tryptic digest of phosphorylase by UPLC technology provides significantly improved resolution, peak capacity, and sensitivity compared to HPLC, allowing the detailed characterization of the protein. HPLC and UPLC chromatograms in analysis of tryptic digest of phosphorylase HPLC and UPLC chromatograms in analysis of tryptic digest of phosphorylase.¹⁹

CONCLUSION:

Decreased column particle size, and column dimensions in UPLC compared to HPLC leads to shortening of analysis time and great savings in solvent consumption thereby decreasing the cost involved. UPLC peaks have decreased noise and better signal to noise ratio. Sharp and narrow peaks with clearer information can be obtained through UPLC when compared to the peaks obtained through HPLC. This technology thus creates a new opportunity for business profitability in highly efficient manner.

CONFLICT OF INTEREST:

The authors have no conflict of interest to declare.

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Received on 31.07.2019 Modified on 24.10.2019

Research J. Pharm. and Tech 2020; 13(3):1570-1574.

DOI: 10.5958/0974-360X.2020.00284.X