Liquid Chromatography with UV Detector Instrument Profile

CHEM5008: Understanding Analytical Instrumentation

Liquid Chromatography with UV detector_ Instrument Profile

Introduction

Liquid chromatography is without a doubt one of the most versatile and popular analytical instruments with a wide range of applications. It is used in diverse industries such as food, pharmaceuticals, biotechnology, medical, forensics and criminology, environment and drug discovery. A typical liquid chromatography (LC) systems are used for High-Performance Liquid Chromatography (HPLC) and Ultra High-Performance Liquid Chromatography (UHPLC) to separate, identify and quantify the mixtures of two or more molecular compounds.

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Detectors in any analytical instrument measure the physicochemical properties of the analytes in the form of a signal.  These measurements depend on the detector system. For example, UV detectors coupled with LC responds to the absorbance, MS detectors will measure based on mass to charge ratio of the analytes and similarly the fluorescence and refractive index by other detectors. Although there are many detectors suitable, a UV detector is commonly in an LC system where analyte molecules are identified based on the measurement of corresponding UV absorbance. This is mainly because of the less complexity of UV detection, lower cost, high sensitivity, and robustness.   

Companies like Agilent, Perkin Elmers, Shimadzu are leading in the LC technologies with advanced series being produced almost every year. However, with newer versions and mature technology, there are still problems associated with it.  These issues can be challenging and time-consuming at the same time. Nonetheless, these issues can still be avoided and preventive measures can be taken while operating the systems. The problems along with its solutions for each LC components will be addressed in the instrumentation part.

The typical design of a modern LC system is demonstrated in the following schematic diagram.

Ref; https://www.waters.com/waters/en_US/How-Does-High-Performance-Liquid-Chromatography-Work%3F/nav.htm?locale=en_US&cid=10049055

The mobile phase (solvents) is stored in the storage bottles. very high pressure for the delivery and mixing of solvent is generated by the pump. An autosampler/ autoinjector will introduce the sample into the mobile phase and the column. The column has packing material effective for the separation of molecular components. This packing material is known as the stationary phase as it is normally placed on the solid silica gels. The detector is wired to the computer data station at the end of the system to see the separated constituents. The mobile phase will exit from detector to the waste and the response generated as a spectral chromatogram is identified and quantified to carry out the analytical study.

Instrumentation

The basic LC system has four major components with specific functions.

1.      The pump

2.      Sampler/ Injector

3.      Column

4.      Detector

Insert the diagram

The pump that is used in modern LC has a basic design of a reciprocating piston. This design comprises of the piston, pump seal, pump head and check valves.  ref

The piston is normally made up of sapphires but stainless steel and graphite can also be used.  The check valves comprise of a rugby ball and sapphire seat which controls the flow rate of the mobile phase corresponding to the pressure.  The piston will forward the liquid into the check valve and the check valves will regulate the flow direction of mobile solvents. There are two check valves. The inlet valves will allow the unidirectional flow of solvent into the piston chamber which in turn will move and allow the liquid to flow into the chamber head. Outlet valves will allow the unidirectional flow of solvent out of the piston head and prevent the backflow of solvent from the column which is under pressure. The pump has to deliver the mobile phase with precision and accuracy. (Dolan, 2016)

Another function of the pump is to mix the solvents. It either mixes the solvents either in constant proportion (isocratic) or in gradient proportion according to the analytical method.  In LC systems, the mobile phase consists of two or more solvents of different ratios. The pump will change the proportion of one of the solvent components over a certain period in gradient proportion. The retention time of the analytes depends on the reproducible flow rate that the pump delivers. The pump also comes with the degasser unit to remove the air dissolved in the solvent.

In the gradient HPLC, two methods of the solvent pump are employed. Binary gradient pump works at high pressure and mixes only two solvent. It can mix the solvent with high precision and accuracy. Thus, it produces shorter delay volume (less sample consumption) which in turn results in faster run time. The other pump is quaternary pumps that mix solvents at low temperature.it will create binary and tertiary gradients and then delivers the mixed solvents into the sampler. Quaternary pumps are flexible compared to the binary pumps as it creates other gradients. the price and maintenance cost are also lower in quaternary pumps as only one pump head is used.  This design approach is common because of well-established solvent delivery. However low-pressure pumps mean less accuracy and reproducibility.

To produce a satisfactory result, the pump should be able to deliver the mobile phase at a high range of pressures.  Newer model of LC systems come with more efficient flexible pumps suitable for the analysis of a wide range of samples.

The autosampler/ injector

In the early days, LC systems has manual injectors where the user has to inject and load the sample manually. Manual injection leads to the overloading of samples which interferes with the separation of compounds. The automatic sampler has now replaced this manual procedure which injects the samples at high accuracy and desired precision. Autosamplers are the mechanism by which the samples are injected into a column for separation. Most commonly autosampler used in almost every LC system today is six-port injection valves. These valves have a fixed stator that is connected to the valve body and a mobile rotor which is held against the stator. the rotor is made up of smooth material such as polymer so that It moves freely during the operation. It is well connected to all the passages of the stator.

HPLCs usually inject sample volumes ranging from 100 nL to 100 uL (W.Dolan, 2001).Two design autosampler is known in the LC system. Loop injection in which the sample can be delivered to a column in either filled or partial loop mode. The volume of the sample in this type of injection varies depending on the size of the loop. Usually, the loop is overfilled with a sample for better reproducibility. This excess sample will be passed to waste port and the appropriate content on the loop will be delivered to the column. This mechanism is known as a filled loop injection. The other option is a partial loop injection in which exactly the measured amount of sample is delivered to the loop and then to column subsequently.  This type of injection is flexible by requires accurate delivery of the sample. As the loop injection causes backflushing of the sample, in case of partial loop injection, ensure correct plumbing– Ferrules, connectors and tubing and the injector port connects to the column.

Another technique used in an autosampler is flowed through which is similar to that of manual injection. The syringe will fill the programmed volume of the sample from the vial, moves to the injection port finally delivering it into the loop. This method of delivering samples can precise and accurate depending on the movement of the stepping motor. It is also known to be highly flexible due to programmable sample volumes.  It also has lower carryovers. carryover is when sample components from previous analysis remain and appear in chromatograms. This happens when molecules retain on the surface of the column and capillary tubes. In flow-through sample injection, there is effective flushing, thus carryover can be avoided.

Column

The column in an LC system is a stationary phase which is responsible for the separation of molecular components. the properties of the column influence the other parts of analytical methods and should be chosen carefully. The resolution and sensitivity of separation depend on the nature of the column used for each analysis. The columns are filled with very tiny particles in a uniform distribution. The separation is based on the chemical and physical properties of molecules such as affinity, polarity, size, etc.  each resulting in separation at the different periods.

the pacing material for the LC column can be of silica, polymers and other gels. Silica is the most popular and commonly used. It has pores on the surface of the gel which provides large surface areas. to achieve high selectivity and better resolution, smaller size particles of porous silicas have been. The current major line is 5μm, but even smaller size 1.5 to 3μm gel is also in use. Ref: https://www.shodex.com/en/kouza/b.html#!
Polymer packing materials are also being used as an alternative. Polyethylene and polypropylene are the most common. Other gel includes natural substances such as dextrin, agar, cellulose, and ceramics.

Examples of the columns used in HPLCs are c18 which are mostly prefeed due to large surface area and high range of hydrophobic separation. C8, C4, and C5 are also used for the separation of macromolecules.

Detector

There are a wide range of detector used in LC system. The detection is based on absorption. Florescence, refractive index, and mass spectrometry. The selectivity, sensitivity and linear range are determined by the type of detector used. In this instrumentation profile, the focus will be on an absorption detector, which is a UV detector.

The basis of UV absorption lies in the photometry of Beer-Lambert Law. The log ratios of the light intensities before and after passing through a certain medium is proportional to the concentration of the analytes, length of the light path and the molar co efficience of the absorbance.

l0l=A=€cd, where A is absorbance, C is concentration, d is the length of light path and € is coefficients of absorbance.

Uv detectors will use photo diodes to capture the dispersed light from monochromator and the spectral absorbance can be identified and quantified. The limitation of UV detectors for LC analysis is that to detect the compound, it must absorb the light between 190-600nm wavelength which is within the range of UV visible region. (Agilent Technologies) Ref Lc handbook

With the control software the detection wavelength can be optimized for different analytes.

The parts of the UV detector comprised of the lamp, the cell, monochromator, and light detecting elements.

Lamp: Deuterium lamp has a spectrum from 190nm to 360nm.  It has a lifetime of about 2000 to 5000 hours. the tungsten lamp in the visible range can emit light around 340nm up to the near-infrared region of 2000nm.  It has a lifetime of about 800 to 2000 hours. The choice of the lamp is restricted when it comes to UV detection. Lamps such as zinc and cadmium could be used but are not common.

Monochromator: The most common monochromator when UV spectrometry is involved is a prism. It is inexpensive and covers wavelength from UV to IR range. However, it has a limitation of nonlinear dispersion which resulted in poor resolution. Now the prisms have been replaced with a grating which is much advanced with linear dispersion and independent of temperature.  It is efficient and generates wavelengths of much higher resolutions

Detector cell: Flow cell determines the optimal resolution and sensitivity. Thus, it should have high pressure, proper volume and optical path length. Flow cell should not be longer than 10% peak volume to have better resolution and avoid unnecessary dispersion. (Meyerb, 2005)

Light detecting Elements: Photodiodes provides high quantum yield, lower noise, excellent linearity, and quick response. Two photodiodes are used in UV detectors. One to capture the light coming from flow cell and others to respond to lights of lamps.

The illustration below shows the flow cell of the UV detector system. After the delivery of analytes into the flow cell, light of wavelength passes through the content in the flow cell which is being captured with photodiodes. This will result in the production of electrical signals. The chromatogram will be generated by measuring these electrical signals against the time-lapse. the intensity of the light is directly proportional to the absorbance and signal. Ref:Crawford scientifi

Troubleshooting

Every LC startup procedure involves the elimination of air bubbles. Check the pump seals which usually warns out during analysis and requires frequent replacements. Flow reproducibility will be affected due to air bubbles and worn out seals.  Some of the doable maintenance includes Manual purging of the systems, replacing inline filter Frites, flushing the column and detector if necessary.

Check valves are prone to fouling by particles

Reference: Introduction to Modern Liquid Chromatography

By Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan third edition, a john wiely and sons  I nc.publications,

Introduction to Modern Liquid Chromatography, 3rd Edition

Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan

ISBN: 978-0-470-16754-0 November 2009 960 Pages