A Comparative Review of Ultra High Performance Liquid Chromatography and High Performance Liquid Chromatography
Table of Contents
List of Figures
List of Tables
1.0 Introduction
1.1 Background and Components of UHPLC
1.2 What is UHPLC?
2.0 Advantages and Disadvantages of UHPLC
2.1 Advantages of UHPLC and HPLC
2.2 Disadvantages
3.0 Differences of UPLC vs. HPLC
3.1 Theoretical Components of UHPLC
4.0 Applications of UHPLC
5.0 Conclusion
6.0 References
List of Abbreviations
BEH – Ethylene bridged hybrid
CDS – Chromatography data system
HPLC – High performance liquid chromatography
HTLC – High temperature liquid column
LC – Liquid chromatography
UHPLC – Ultra performance liquid chromatography
UFHPLC – Ultra fast high performance liquid chromatography
VHPLC – Very high performance liquid chromatography
Figure 1. Schematic flow channel diagram of the basic components of a UHPLC or an HPLC system (Minic, 2018).
Figure 2. Waters ACQUITY UPLC Waters ZMD system components identified (Minic, 2018).
Figure 3. Isocratic mode of the pumping system (Minic, 2018) .
Figure 4. Graphical representation of decreasing particle diameter to increase separation of compounds (Minic, 2018).
Figure 5. Graphical representation of the variety of different fields that utilize the applications of ultra high performance liquid chromatography (Alexander, 2009).
Table 1. Summary of Comparison between HPLC and UHPLC.
1.1 Background and Components of UHPLC
High Performance Liquid Chromatography (HPLC) is a chromatographic technique, in which “chromato” means colour and “graphy” means writing. Hence, HPLC is a method of colour writing (MacKintosh, 2006). This colour writing is displayed in a chromatogram, which displays colours in a graph to show the separation of two or more compounds. HPLC was pioneered by Christian Fridrich Schönbein who first experimented with moving substances through a filter paper (Chromatography Today, 2015). Mikhail Semyonovich Tsvest then began to separate plant extracts in Calcium carbonate columns (Ettre, 2003). Essentially, high performance liquid chromatography is the separation of compounds on a stationary phase column. The basic components of the HPLC system consist of the following: (Figure 1)
Figure 1. Schematic flow channel diagram of the basic components of a UHPLC or an HPLC system (Minic, 2018).
1) Eluent or Mobile Phase
A solvent which runs continuously through the system and pushes the sample through the column. The solvent is contained in the reservoir which is located at a higher elevation than the pump in order to maintain a slight positive head pressure on the pump and the inlet. The pumping system filters the solvents to avoid any damage to the instrument.
2) Auto sampler / Sample injection unit
An auto-sampler stores the samples in vials or plates and these are stored under a temperature controlled environment. When the sample is ready for separation, the data system signals the auto sample to eject the sample. This allows a user to perform multiple sequences of runs.
3) Column
A column is packed with stationary phase that separates the sample. A non-polar silica phase is most common for reversed phase HPLC. Therefore, the longer the column, the higher the efficiency and resolution. If the column is shorter, it has a faster separation. If the column has a larger diameter, then the loading capacity is greater. If the column consists of a narrow diameter, it results in a greater mass sensitivity. Overall, the factor of column length and diameter influences the results obtained by the HPLC.
4) Detectors
There is a placement of one or more detectors in series which receives the result of the sample separation from the column and monitors the physical properties and changes of the sample as it elutes. These detectors include UV-VIS absorbance detectors, photodiode array-type UV-VIS absorbance detector, fluorescence detector, refractive index detector, electrical conductivity detector, electrochemical detector, and mass spectrometers.
5) Chromatography Data System (CDS) / Data Processor
The data system translates the signal from the detector to the chromatographic spectrum which results in qualitative and quantitative data about the sample. Qualitative data gives the components of the mixture for a sample of interest. Qualitative data analysis is identified based on retention time, and the acquisition of spectra is used primarily with UV spectra and MS spectras. After the qualitative analysis acquisitions, the sample is transferred to the other analytical instruments after preparative separations. Quantitative data results in the answer to the concentration of these mixtures within the sample of interest. The data system allows complete control of the pump, auto sampler, and the detector. All of the instrument’s parameters, automated run sequences, and data collections, can be controlled by the data system. 
Figure 2. Waters ACQUITY UPLC Waters ZMD system components identified (Alexander, 2009).
Ultra high performance liquid chromatography (UHPLC) is a technique that could achieve increased separation efficiency, improved resolution and shorter analysis times, and also lower operation costs. It does this by using HPLC columns with mean particle size diameter that is less than 2 µm along with the new commercially available instrumentation capable of driving the analyte through the column at subsequently higher pressures. This allows optimal linear velocities to be reached for smaller particle size columns.
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Advancements in HPLC have been growing within the past 15 years. These advancements include the improvements of the stationary phases which have lead to higher separation efficiency results, faster analysis, and better resolution outcomes. HPLC was the technique of choice for a long period of time. However, the use of HPLC had some limitations such as limited capabilities of efficiency and speed. These problems were the main reasons UHPLC was developed to approach a highly efficient and fast liquid chromatographic approach. There are four important factors for performing fast and highly efficient liquid chromatographic separations: UHPLC, core shell column, monolith column, and high temperature liquid column (HTLC). This report will focus on UHPLC. The term ultra high performance liquid chromatography uses other acronyms such as the following: UHPLC, UPLC, VHPLC (very high performance liquids chromatography) or UFHPLC (ultra fast high performance liquid chromatography). Among these acronyms, UPLC is a discouraged term because it is trademarked by Waters Cooperation. In contrast, UHPLC is the most generic term commonly used (Nováková, Svoboda, & Pavlík, 2017). In this report, for sake of convenience and proper scientific terminology, it will be referred as UHPLC.
UHPLC was used in replacements of conventional HPLC and is one of the most widely used fast liquid chromatographic approaches. This report will focus on the key aspects of UHPLC such as its advantages and disadvantages, theoretical aspects, and applications for drug discovery. The main objective of this UHPLC review is to understand the concept of UHPLC and identify its incredibly efficient way of separating small molecules, within minutes, resulting in highly efficient data.
2.0 Advantages and Disadvantages of UHPLC
2.1 Advantages of UHPLC and HPLC
UHPLC is a new class of separation science and it provides improved resolution, speed and sensitivity. It is suitable for chromatographic application in general and is appropriate for developing new methods and improving existing methods. UHPLC is similar to HPLC but with very high pressures and columns withstanding very small particle sizes. The main advantages of UHPLC are its high resolution, speed and sensitivity. Using the Van Deemter equation these terms will be focused in section 3.1 of this report. UHPLC is known to give results nine times faster than conventional methods and produce two times the resolution and three times the sensitivity of HPLC systems (Nováková, Svoboda, & Pavlík, 2017). The utility of these instruments are currently rising in various different applications due to its quick and easy instrument handling. This instrument is customer friendly with high sensitivity results. In addition, neon particles can also be easily separated. In the 1890s, detecting very small amounts of gases was very difficult. The instruments used during those times were not able to capture the small fractions of a millilitre of gas. In 2018, it is now very easy to detect these small amounts of gases due to spectroscopy methods advancing rapidly. It is important to convert the use of HPLC to UHPLC methods due to faster result acquisition, greater information acquisition and the use of more robust methods. UHPLC systems are efficient with analyzing multiple samples which increases the productivity of the user.
HPLC consists of high separation capacity which enables the analysis of multiple components. HPLC also consists of high quantitative capability and reproducibility. Generally it produces high sensitivity and undergoes a low sample consumption. HPLC uses easy preparation and purification of samples (Nováková, Matysová, & Solich, 2006). However, there are many types of HPLC techniques based on principle of separation, elution, scale of operation and analysis. We can use absorption chromatography, ion exchange chromatography, size exclusion, affinity chromatography and choral phase chromatography for techniques involving separation of compounds. Isocratic separation or gradient separations can be used based on elution techniques in the HPLC (North America, 2013). Isocratic separation is used when the compound’s polarity and hydrophobicity varies widely. Isocratic separations produce poor resolution, early elution of peaks, and increased peak width of later eluted compounds due to peak dispersion (Figure 3). This often results in unnecessarily long analyses. Whereas gradient HPLC, on the other hand, provides better peak shapes and elutes analyte in a reasonable time frame (North America, 2013). Gradients used in reverse phase HPLC usually involve mixing of solvents to achieve steady increase in the organic solvents over the course of analysis, serving to increase the solution strength of the eluting over time. (Nováková, Svoboda, & Pavlík, 2017)
Figure 3. Isocratic mode of the pumping system (Minic, 2018).
Analytical or preparative HPLC techniques can be used based on scale of operation. Lastly, either qualitative or quantitative analysis can be used based on analysis of HPLC technique.
UHPLC uses high pressures for separating samples. Therefore, high pressures require higher maintenance to the instrument. Additionally, high-pressures also reduce the life of the columns. Columns used in the UHPLC systems have concrete specifications. For example, an UHPLC column has an ethylene bridged hybrid (BEH) structure and has high mechanical strength, efficiency, and high pH stability and peak shape for bases. UHPLC columns are usually packed with C8, C18, Phenyl, and HILIC. The pH can range from 1-12 and can withhold a maximum pressure of 15,000 psi. The particle size of the column is 1.7 µm with a pore diameter of 130A/ 0.7 mL/g. Lastly, it contains a surface area of 185 m2/g. (Nováková, Svoboda, & Pavlík, 2017) In short, it is expensive to maintain an UHPLC instrument because it requires special care and cost to keep the instrument running smoothly. In addition, the stationary phase and mobile phases, which are less than 2 µm, are non-regenerable and thus have limited use. Again, this is another costly factor.
In summary, there are many advantages of using UHPLC which outweigh the disadvantages when it comes to acquisition of results.
3.0 Differences of UPLC vs. HPLC
High performance liquid chromatography was used in the early 1970s.A HPLC system consist of a particle size ranging from 10, 5, and 3.5µm particles. It also consists of a column diameter of 4.6 x 250 mm, 2.1 x 150 mm, and 1.0 x 150 mm. The maximum pressure of the system can reach up to 6,000 psi, but generally uses 1,000 – 3,000 psi. It takes approximately 45 minutes for gradient analysis. (Table 1)
In comparison, the UHPLC was developed in 2004 by Waters Corporation. UHPLC uses very small particle size of 1.5 µm. The maximum pressure of the instrument reaches 15,000 psi with a general range of 200 to 14,000 psi. UHPLC uses gradient analysis and takes only 4.5 minutes. The importance of gradient analysis has been explained in Section 2.1.
|
Comparing HPLC to UHPLC |
||
|
HPLC High performance LC |
UHPLC Ultra High Performance LC |
|
|
Developed |
1970 |
2004 by Waters® |
|
Particle Sizes |
3.5 , 5 and 10 µm |
1.7 µm |
|
Column Sizes |
4.6mm x 250 mm 2.1mm x 150 mm 1.0mm x 150 mm |
2.1mm x 150 mm 1.0mm x 50 mm |
|
Maximum Pressure |
6,000 psi |
15,000 psi |
|
Time for analysis |
45 min |
4.5 min |
Table 1. Summary of Comparison between HPLC and UHPLC (Cielecka-Piontek, Zalewski, Jelińska, & Garbacki, 2013).
3.1 Theoretical Components of UHPLC
It has been previously emphasized that the UHPLC is advantageous due to its speed, resolution and sensitivity. However, it also consists of additional advantages of injection repeatability, gradient repeatability, column to column repeatability, instrument versatility and reduction of solvent usage and waste minimization. To understand the theoretical aspect of the speed, resolution, and sensitivity, it is important to review the components of the Van Deemter equation (Equation 1)(Nováková, Svoboda, & Pavlík, 2017). The greater the number of theoretical plates (N), the greater the separation of the compounds in a mixture. Therefore, to calculate N, equation 2 displays the relationship of column length and particle size. Overall, it is ideal to decrease particle size, dp, to obtain higher efficiency according to the relationship of the equation. Furthermore, as particle diameter decreases from 5µm to 1.7 µm the separation efficiency and resolution increase, displayed in Figure 4. 
Equation (1)
H is plate height, λ is particle shape (with regard to the packing), dp is particle diameter, γ, ω, and R are constants, Dm is the diffusion coefficient of the mobile phase, dc is the capillary diameter, df is the film thickness, Ds is the diffusion coefficient of the stationary phase, u is the linear velocity (Cielecka-Piontek, Zalewski, Jelińska, & Garbacki, 2013).
Equation (2) N = L / dp
where, N= number of theoretical plates, L = column length, dp = particle size. (Cielecka-Piontek, Zalewski, Jelińska, & Garbacki, 2013)
Additionally, other factors such as pressure are important for the effects of pressure, column length, and particle size in liquid chromatography. This relationship uses the pressure equation 3.
Equation (3)
Figure 4. Graphical representation of decreasing particle diameter to increase separation of compounds (Minic, 2018).
The advantage of sub-2µm particles sizes play a role in the speed of the UHPLC instrument. With a particle size changing from 5µm dp to1.7 µm dp, the speed is increased by nine times. With a particle size changing from 3.5 µm dp to 1.7 µm dp, the speed is only increased four times. In summary, each of the variable in the van deemter equation acts upon the instrument to produce plate height in liquid chromatography.
In the pharmaceuticals industry, UHPLC is an instrument that is now recommended in every industry due to its multiple analysis capability. Application of UHPLC range from “injection of a poly-drug reference standard and whole body extract to separation and identification of amphetamine, methamphetamine, ephedrine, pseudoephedrine, phentermine, MDA, MDMA, MDEA and ketamine being assessed in less than 3 minutes” (Nováková, Matysová, & Solich, 2006) . In the pharmaceutical industry, the instrument is used primarily for quality control to monitor the compliance standard of drugs regulated. In Research and Development (R&D), it is used to identify the structure of the molecule or to determine the components of the mixture (Chawla & Ranjan, 2016). The application in UHPLC do not solely benefit the pharmaceutical industry, Figure 5; it is also very useful to the following: biogenic substances, such as sugars, lipids, nucleic acids, amino acids, protein, peptides, steroids and amines; medical products, such as drugs and antibiotics; food products, such as vitamins, food additives, sugars, organic acids and amino acids; environmental samples, such as inorganic ions and hazardous organic substances; and organic industrial products, such as synthesis polymers, additives, and surfactants (Nováková, Matysová, & Solich, 2006). Furthermore, HPLC can be used specifically in applications such as water purification, ligand-exchange chromatography, ion-exchange chromatography of protein and many more (Nováková, Matysová, & Solich, 2006). HPLC analysis is widely used in clinical diagnosis and in the health industry for scientific research, to develop standards by government, and for separation of similar molecules. The applications of UHPLC and HPLC are increasing due to the instrument coupling with detectors such as mass spectroscopy.
Figure 5. Graphical representation of the variety of different fields that utilize the applications of ultra high performance liquid chromatography (Alexander, 2009).
UHPLC has been widely adopted to various fields of applications due to its easy user interface and higher results acquisition in comparison to the conventional HPLC methods. Previously, the field of chromatography had started with gravity based separation techniques. Then, in 1890’s the field of chromatography has been escalated with the invention of HPLC. Since then, HPLC instruments have been advancing based upon mobile phase, particle sizes, column packing and column diameters. In 2004, the development of UHLP has broadened the applications of liquid chromatography. At the beginning, many industries and researchers were hesitant in the usage of UHPLC due to its costs and lack of experienced users. After an influx of literature publications using UHPLC to result in high resolution, efficiency and speed, the use of UHPLC has been popularized. The unique factor of UHPLC is its ability to run the experiments at maximum pressure of 15,000 psi. In contrast, HPLC can only run up to 6,000 psi. The high pressure in combination with lower particle sizes of less than 3µm have increased the run time by 4-9 times fold. The advantages of UHPLC are its acquisition of results in rapid time and multiple analysis performance. The disadvantaged of the UHPLC include the maintain and cost of the instrument and specific column requirements. Due to UHPLC running at high pressures, it requires specialized components such as special UHPLC columns with specific requirements, and unlike HPLC, UHPLC requires regular maintenance.
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Applications of UHPLC are of great importance in the field of pharmaceutical industry due to its multiple analysis feature. The users of UHPLC can analyze up to 40 different compounds in 45 minutes whereas the HPLC can only analyze one compound in 45 minutes. The screening of drugs by UHPLC is performed for quality assurance purposes. The health care fields use UHPLC and HPLC to meet the standard of the government drug regulations (Chawla & Ranjan, 2016). It is an important application in the modern era of science research. Without the use of UHPLC, it can be difficult for an industry to function time-efficiently. Therefore, there is strong evidence for converting from HPLC methods to UHPLC. Overall, this report summarizes the terminology of UHPLC, its advantages and disadvantages, and its applications in variety of different fields.
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