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HPLC Basics
1.0 The HPLC
In the 1960s, column chromatography LC with its glass columns (Fig. 1) in the low pressure range was developed into HPLC with metal columns (Fig. 2) in the high pressure range.
HPLC = High Performance Liquid Chromatography
LC = Liquid Chromatography
Just like LC, HPLC is used to separate substances from a sample, also called purification or isolation, however, the separation is completed much faster with HPLC than with LC. The prerequisite for liquid chromatography is that the sample is dissolved in a solvent.
The purification takes place in a separation column between a stationary and a mobile phase. In this type of chromatography, the stationary phase is a granular material with very small, porous particles in a separation column (Fig. 3). While the mobile phase is a solvent or solvent mixture that is pressed through the separation column at high pressure. Via a valve with a connected sample loop (Fig. 4), a small tube or a capillary made of stainless steel, the sample is injected into the flow of the mobile phase from the pump into the separation column using a syringe.
Mobile phase also called eluent, flow agent or solvent.
The individual components of the sample then migrate through the column at unequal velocities as they are held back to different degrees by interactions with the stationary phase. After exiting the column, the individual substances are recognized by a suitable detector and transferred as a signal to the HPLC software in the computer. At the end of this process / run, a chromatogram is obtained on the computer in the HPLC software (Fig. 5) which can be used to identify and quantify the various substances.
By this time, HPLC has developed into a universally applicable method, so that it is used in almost all areas of chemistry, biochemistry and pharmacy.
Typical applications of HPLC are:
- Analysis of medical substances
- Analysis of synthetic polymers
- Analysis of pollutants in environmental analysis
- Determination of active ingredients in biological matrices
- Isolation of valuable products.
- Purity and product control of industrial products and fine chemicals
- Separation and purification of biopolymers such as enzymes and nucleic acids
[Fig. 1]
[Fig. 2]
[Fig. 3]
[Fig. 4]
[Fig. 5]
2.0 HPLC Versions
The main distinction is made between isocratic HPLC and gradient HPLC.
2.1 In isocratic HPLC, only one eluent composition is used during the entire run. This means that the interaction between the mobile and stationary phase is constant over the complete separation period. You can use it to separate similar substances. This type of implementation is particularly popular in the routine and should, if possible, be aimed at for all separation tasks. Another advantage is that only one pump is required to deliver the eluent for this method.
2.2 With a gradient HPLC, the composition of the eluent changes continuously over time. In this way, a constant change in the interaction between the eluent and the stationary phase is achieved. The sample substances are displaced quicker from the exchange sites on the stationary phase. It is thus also possible to separate substance components of very different polarity within an acceptable time. Since the composition of the eluent at the beginning of a separation differs from that at the end, it must always be ensured that the system is in equilibrium at the beginning of a separation (= equilibration). The mixing of the solvent can be carried out in various ways depending on the equipment used.
2.2.1 Low Pressure Gradients
The different eluents are mixed by a proportioning valve prior to the pump head and the mixture is conveyed through the system via the pump. This is referred to as a low pressure gradient. Mixing takes place on the low-pressure side (normal pressure) of the system, i.e. upstream of the pump.
2.2.2 High Pressure Gradients
Each eluent is delivered by a pump and mixing takes place on the pressure side of the system. A mixing chamber is used for this. Such a system is called high pressure gradient HPLC. Pressure builds up when the eluent is conveyed through the HPLC system. The main part is caused by the column. Depending on the separating material, its grain size and shape, the column length and the eluent used (different viscosity), pressures of up to approx. 300 bar and more result.
3.0 Structure of Some HPLC Systems
The HPLC usually consists of five main devices: pump, injection unit, separation column, detector with evaluation software and fraction collector. HPLC uses separation particles with grain sizes of 3 to 20 µm, thus achieving a high number of separation stages, yet at the same time the transport of the mobile phase through the separation column requires overcoming a relatively high counterpressure. All parts must be joined together with as little dead volume as possible and be pressure-stable.
Analytical HPLC
Analytical HPLC usually consists of one HPLC pump with a flow range of 0.01-10 ml/min, a UV detector, an autosampler and a PC with analytical HPLC software.
Semi-Preparative HPLC
Semi-preparative HPLC usually consists of one HPLC pump with a flow range of 0.01 – 50 ml/min, a separation column, a UV detector, a fraction collector and a PC with preparative HPLC software.
Preparative HPLC
Preparative HPLC usually consists of two HPLC pumps with a flow range of 0.1 – 100 ml/min each, a separation column, a UV-Vis detector, an autosampler / fraction collector and a PC with preparative HPLC software.
4.0 HPLC Pumps
4.1 The HPLC piston pump is an electromechanical device for pumping the mobile phase (eluent, solvent) under high pressure, up to approx. 600 bar. In the pump head, a piston is moved back and forth through seals, whereby the eluent is sucked into the piston chamber via the inlet valve and is pressed towards the separation column via the outlet valve (Fig. 6). By using several pump heads, pulsation-free delivery of the solvent can take place, because while one piston draws in via the inlet valve, the other piston expels the previously drawn-in solvent via the outlet valve.
[Fig. 6]
4.2 The HPLC membrane pump, in contrary to the piston pump that conveys the eluent via a piston, transports the eluent via the movement of a membrane. The membrane is set in motion by the back and forth movements of a piston in oil, thereby sucking in and ejecting the eluent. Ball valves also regulate the path of the liquid here.
This type of pump is mainly used in industrial preparative HPLC systems.
5.0 The Sample Application
The sample can be applied manually with a syringe via a valve with a sample loop (Fig. 7) or automatically via an autosampler (Fig. 8). The sample must be injected into the pressurized line of the system under ambient pressure.
5.1 For manual sample application, the sample is drawn into a syringe and initially injected without pressure into a sample loop that is connected via a multi-way valve. By switching the valve to the pressure side, the eluent flow is guided through the sample loop in the direction of the separation column.
5.2 For automatic sample application, the sample is taken from vessels that are located in an electronically controlled device, for example in an autosampler. The samples then also reach the column via an electrically operated valve.
Some autosamplers offer the possibility of sample preparation. This includes automated dilution and mixing of the sample and pre-column derivatization. With the appropriate HPLC software, the devices automatically dilute highly concentrated samples so that the highest level of analytical accuracy is achieved after just a few injections and overloading of the column is prevented. If the concentration is outside the calibrated range, these over-range samples are also diluted so that there is no need to analyze them again. In order to shorten the analysis time, some devices work with the sample overlap function. While one sample is still being separated, the next one is already being prepared. Mixing with an internal standard, adding reagents or preparing standard solutions and calibrations are further possible functions of the autosampler.
[Fig. 7]
[Fig. 8]
6.0 HPLC Columns / HPLC Separation Columns
The HPLC columns usually consist of a stainless steel tube and screw caps at both ends with a connection for a capillary on one side as an inlet and as an outlet on the other side.The HPLC columns are filled with the stationary phase, a very fine-grained, porous powder (3, 5, 8 or 10 µm grain size) under mechanical pressure, without empty space. The smaller the grain size, the higher the separation efficiency. The column material is always selected according to the separation task to be completed. Probably the most frequently used HPLC column is a “C18 column”. In this case, the stationary phase is a chemically modified silica gel with C18 chains built up on its surface. To ensure good reproducibility of the results, the HPLC column should be operated at a constant temperature. In the HPLC column, the sample substances are adsorbed on the stationary phase and separated by adsorption / desorption processes.
The analytical separation column often has an internal diameter of 2 to 5 mm and a length of up to 250 mm and is designed for a maximum flow of approx. 2 ml/min.
The semi-preparative separation column with an internal diameter of 10 – 50 mm and a length of 250 mm is suitable for a maximum flow of approx. 100 ml/min.
The preparative separation columns with an internal diameter from 75 mm and a length of up to 1000 mm are used with a flow from 100 ml/min.
The precolumn is mounted directly in front of the separation column to prevent contamination. The precolumn is smaller and almost always contains the same column material as the separation column.
[Fig. 9]
6.1 The Column Packing Station “Pack columns yourself”
If you want to be flexible with the column material, you can use a column packing unit – also called a column packing station.
Thanks to static or dynamic axial compression, all common column materials can be packed reproducibly in the required bed length by the user.
Due to the simple packing process, the HPLC column can be quickly adapted to requirements and there is no need for a selection of expensive ready-made columns. The carrier material can be packed with high pressures through a frit that is firmly connected to the movable piston. This ensures an even distribution of the substance over the entire cross-section of the column and an optimal peak shape.
[Fig. 10]
Compression of the column bed can be achieved in two ways. Either with space-saving manual hydraulics or with pneumatically driven hydraulics for static and dynamic axial compression. With static compression, the HPLC column can be removed from the packing station. This means that several HPLC columns can be produced with one packing station. On the other hand, with dynamic axial compression, the HPLC column remains in the packing station and the column bed is automatically kept under pressure so that there is no free space at the column inlet that could affect the separation performance. If the separation efficiency of the removed HPLC column declines due to the resulting free space, re-pressing is sufficient to achieve the best separation results again. Furthermore, with a column packing station, different bed lengths can be realized with one column tube and, as an option, temperature control over the entire bed length is possible when using a column tube with thermal jacket.
The Packing Process:
The bottom flange is mounted on the column tube. The slurry (emulsion of carrier material and a suitable solvent such as isopropanol) is poured into the column tube. The slurry (column material) is pressed hydraulically, the solvent in the slurry is displaced from the column tube, so that a homogeneous solid packing is created from the packing material. The piston is fixed (static compression) or automatically pressed (dynamic axial compression).
[Fig. 11]
7.0 HPLC Detectors
The HPLC detectors job is to record the sample molecules separated in the separation column and strongly diluted in the mobile phase and to pass this information on to the HPLC software in the computer.
7.1 The UV detector is currently the most commonly used detector in HPLC. Here, UV light with a certain wavelength is radiated from a deuterium or quartz lamp in the detector through a flow cell. It is captured on a photodiode behind it (Fig. 12) and passed on to the HPLC software as an electrical signal. With a photo measuring cell on the flow cell, the UV detector constantly measures the extinction of the light after penetrating the solvent with the sample molecules, which flows through two panes of glass with constant distance. The attenuation of the radiation (extinction) and thus the change in the electrical signal, therefore only changes due to the concentration and the type of medium. Since the eluent and sample absorb the light differently, the result is a changing electrical signal. These changes are displayed as substance peaks in the HPLC software. The chromatogram is usually composed of several peaks.
Detection limit approx. 0.3 ng/ml
[Fig. 12]
7.2 The Diode-Array Detector (DAD) – also called Photo-Diode-Array-Detector (PDA), measures, just like the UV detector, light absorption by the mobile phase in the ultraviolet or visual wavelength range. However, the conversion into the electrical measurement signal is not done by a photo measuring cell, but mostly by 512 or 1024 photo diodes which are arranged in a square area, the diode array. This makes it possible to capture the entire spectral range, i.e. several wavelengths at the same time.
Detection limit approx. 0.3 ng/ml
7.3 The Refractive Index detector (=RI) is less sensitive than the UV detector, but it can detect substances that show no UV absorption. It measures the difference in the refractive index between pure solvent and solvent mixed with a sample. However, the RI detector always requires a reference channel with pure mobile phase for measurement. The more the refractive indices of the mobile phase and reference substance differ, the greater the signal to the chromatogram for the substance peak. It is mainly used in polymer chemistry and sugar analysis.
Detection limit approx. 0.7 µg/ml
7.4 The fluorescence detector (FLD = Fluorescence Detector) is up to 1000 times more sensitive than the UV detector. It uses the property of fluorescent substances to detect them.
Detection limit approx. 0.8 pg/ml
7.5 The electrochemical detector (ECD = Electron Capture Detector) oxidizes or reduces the substance components by applying a potential. The change in potential indicates the presence of a substance component. It is mainly used in clinical chemistry.
Detection limit approx. 1.0 pg/ml
7.6 The light scattering detector (ELSD = Evaporative Light Scattering Detector) is used when substance classes such as lipids, carbohydrates, polymers, surfactants or amino acids are not or only slightly UV or VIS-active. It is very well suited as a universal detector in HPLC and GPC because it uses a physically different detection principle. The eluent stream is nebulized with compressed air / nitrogen and evaporated in a drying unit made of glass. In the resulting aerosol particles, the analytes are measured in a mass-selective manner by means of light scattering. In contrast to the RI detector and electrochemical detector (ECD), this detection principle allows the use of eluent gradients. The light scattering technology enables maximum sensitivity and drift-free detection without cumbersome setting procedures.
Detection limit approx. 5 ng/sample component
8.0 Fraction Collector
A fraction collector ensures you don’t lose any valuable substances after successful separation. In addition to drop- and volume-dependent collection, fractionation over time segments, time windows and / or peak detection is particularly helpful. Optional valves ensure contamination-free collection in the individual vessels. A wide variety of rack formats from microtiter plates to large volumes usually allow a wide range of applications. Programs can often be saved and called up automatically via the HPLC software. The software enables an exact assignment of your fractions to the collection events recorded in the chromatogram via an integrated sample tracking as well as the documentation of the fractionations .
[Fig. 13]
9.0 HPLC Software
Usually a computer with HPLC software (Fig. 14) is used to control the system and evaluate the measurement results. For qualitative analysis, all peaks contained in your sample are simply separated (fractionation). For quantitative analysis, the exact concentration or amount of substances present in your sample has to be known. Consequently the evaluation can be carried out via the area of the peaks.
[Fig. 14]
9.1 The Chromatogram
Depending on the type of detector, a differential chromatogram (Fig. 15) or an integral chromatogram is obtained, whereby the integral chromatogram is very rare. The portion of the chromatogram recorded by the detector when only the mobile phase is eluted is called the baseline. The detector deflection for an analyte is called a peak. If the separation is incomplete, several substances can be accumulated within one peak. The interpolation of the baseline between the beginning and the end of the peak is called the base of the peak. The peak area is enclosed by the entire signal of the peak. The peak maximum represents the extreme value of the detector signal, the peak height represents the distance between the maximum and the base line of the peak.
[Fig. 15]
We hope this information is interesting for you and perhaps also helpful for your work.
With kind regards,
your O-CP GmbH
Source – Handbook of HPLC: Handbuch der HPLC, K.K. Unger, GIT Verlag