theory of hplc - chromacademy · instrumentation of hplc ... to describe the individual components...

Instrumentation of HPLC

Autosamplers

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Aims and Objectives

Aims and Objectives

Aims

To explain the purpose of injectors and autosamplers within the HPLC system

To describe the principles behind which sample injection systems work

To describe the individual components of an injection valve

To highlight correct and incorrect ways to use loop injectors in manual injection mode

To describe the major components and operating principles of a range of typical autosampler devices

To outline common autosampler problems and to introduce some troubleshooting principles

To outline how carry-over may occur within the autosampler and how to minimize it Objectives At the end of this Section you should be able to:

Describe the purpose of sample introduction systems within HPLC

Demonstrate an understanding of the components and operating principles of injection valves

Select correct sample volumes to be aspirated into loop injectors to optimise quantitative accuracy

Describe the main operating principles of a range of typical autosampler devices

Recognise advantages, disadvantages and potential problems of each type of autosampler

Outline strategies for reducing carry-over in autosampler devices

© Crawford Scientific www.chromacademy.com

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Content Introduction 3 Injection Valves 4 Rotor Seals 6 Troubleshooting 8 Manual Injection Systems 9 Manual Injection Complete and Partial Loop Filling 11 Autosamplers 13 Pull to Fill Auto Samplers 15 Push to Fill Auto Samplers 20 Integral Loop Auto Samplers 24 Autosampler Contamination 27

Carry Over 27 System Contamination 27 Sample Contamination 28


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Introduction The injection system is positioned after the pump head, and is therefore located on the high pressure side of the solvent delivery system. The injection of a sample at atmospheric pressure into the system, at high pressure, represents a critical step in the chromatographic process. Sample injection valves, or switching valves, are used to introduce reproducible amounts of sample into the HPLC eluent stream without causing changes in pressure or flow. Injecting directly into the high pressure flow of mobile phase eliminates the requirement to stop the mobile phase flow, effectively minimising any disturbance in the chromatographic baseline by allowing an essentially pulse-free change in the direction of the mobile phase flow through the autosampler valve.

The injector / autosampler


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The purpose of the injection system is to introduce a volume of sample solvent into the mobile phase flow without broadening the chromatographic band or introducing excessive amounts of air. Injection Valves The valve is the heart of the autosampler system and operates by controlling the flow of eluent through the autosampler device to allow loop filling in one configuration and to sweep the loop contents onto the HPLC in the other configuration. Of course, more sophisticated valves can also be used for applications where perhaps two different sample loops are required or where analyte needs to be directed onto different analytical columns etc. Valve injection allows the rapid, reproducible and essentially operator independent delivery of a wide range of sample volumes (e.g. from 60nl up to several millilitres), at pressures up to 7000psi with less than 0.2% error. High performance valves provide extra column band broadening characteristics comparable or superior to that of syringe injections. Manually operated valves are only moderately expensive and automated versions can be obtained at somewhat higher costs. A further advantage of using sampling valves is that they can be located within a temperature-controlled oven for use with samples that require analysis at either elevated or lowered temperatures. The most common valves can be obtained in either 4 or 6 port configurations for use in either manual or automated mode.

Important Injection System

The injector is located on the high pressure side of the pump

Switching valves allow effective sample introduction without interrupting the flow or altering the system pressure

Components of the switching valve include

A needle or syringe to pierce the vial septum

A metering device to measure the aspirated amount of sample liquid

A loop or holding device to retain the sample prior to injection

A valve which is used to alter the hydraulic path of the eluent through the device in order to affect direct injection of the sample plug into the eluent stream under pressure


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Injection valves

The operating conditions of the LC system can influence the choice of rotor seal that must be made. Vespel 211 is the most commonly used material. It is a polyimide resin containing 15% graphite. Vespel rotor seals will tolerate solutions that have a pH up to 10. If the system is operating at a pH above 10 (or at very low acidic pH) it is recommended that either PEEK of Tefzel rotor seals are used. TEFZEL® known as ETFE (a copolymer of Ethylene-tetraflouroethylene) is a rugged fluorine based thermoplastic which resists virtually every known chemical, from acids and alkalis, to solvents and organics. It's mechanically tough, chemically inert and offers a wide range of temperature resistant ranging from cryogenic to 120º C.


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Rotor Seals Valve injectors work by controlling the flow of liquid through components that allow the sample ‘loop’ to be either isolated from, or included in, the mobile phase flow. When isolated from the flow the loop may be filled and when required the mobile phase flow can be directed in order to flush the loop contents into the system. The injector is able to control mobile phase flow using a component with etched flow paths (the rotor seal), which are compressed against a smooth face stator using a compression spring mechanism. The stator allows mobile phase to flow into and out of the injection head and the direction of flow depends on which paths are connected by the channels in the rotor seal.

Rotor seal with two flow paths from a four port injection device (used typically for manual

injection)

Rotor seal with three flow paths from a six port injection device (used typically for

autosampler injection) It is necessary to divert the flow of mobile phase away from the sampling system (i.e. the syringe) when aspirating the sample loop prior to injection. This is achieved using the injection valve containing a Rotor Seal. The interface between the HPLC capillaries and the stator is known as the Stator Face.


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Situated directly beneath the Stator Face is the Stator itself which is used to transport eluent or other liquids from various ports on the valve head to the channels on the Rotor Seal. Essentially the Rotor Seal ‘joins’ liquid paths via the stator. As Rotor Seal turns against the Stator under high pressure it is important that the contact between these components is leak tight, flat and even. This is achieved by the use internal Springs and Washers, further more Retaining Bolts that hold the injection valve components in place are tightened with equal torque. Although most modern valves are self-levelling, older models use ‘set-screws’ which must be adjusted to ensure the stator face is level with the valve body –this is usually checked with the aid of ‘feeler gauges’. On automated systems the Rotor Seal is moved using an electronic or pneumatic motor.

Holding Screws: Hold all the injection valve components in place. There are tightened with equal torque. This helps ensure the contact between the rotor seal and stator is even, ensuring a liquid tight seal. This also helps to prevent rapid degradation and uneven ware on the rotor seal that will lead to leaks and poor injection volume reproducibility. NOTE, some Rheodyne devices also have small ‘set screws’ which need to be adjusted in order to ensure that the stator and rotor seals are properly aligned. Stator face: The stainless steel valve head into which the various pieces of tubing are screwed to allow mobile phase to flow into and out of the valve paths. The sample loop and waste tubing will also be connected to the stator face. Stator: A ceramic component with a smooth face plate through which small holes are drilled. These holes bring liquid from the various points on the valve head (stator face) and feed through to the channels on the rotor seal –which joins flow paths together via the channels etched into it. The stator forms a liquid tight seal with the rotor seal when the valve is screwed together.


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Isolation seal: Isolates the shaft from the valve housing to allow the rotor seal to turn with the shaft, as well as protecting the stepper motor mechanism for any potential leaks from the valve body. Rotor seal: Using the channels etched into the surface, different hydraulic paths are connected as the rotor seal turns. The mobile phase, or will by pass the sample loop during the sample aspiration (loading) phase of the injection. Motor: A motor is used to automatically turn a shaft to which the rotor seal is directly connected via pins. This allows different hydraulic flow paths to be selected for the eluent during the sample loading and injection phases.

Troubleshooting The rotor seal is constantly rotating against the ceramic stator under a great deal of torque. Over time the channels on the rotor seal will start to widen. If particulate materials are trapped between the stator face and rotor seal, as the two turn, a scratch may develop between the two channels. The scratch may eventually develop into a ‘cross port scratch’ which ultimately results in sample or eluent leaking out of the device into the waste port during the injection or analysis phase of the injection. The poor mass transfer of the wider sample plug will ultimately result in broad chromatographic peaks and poor peak area (or height) reproducibility.


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Manual Injection Systems Manual sample injectors for HPLC transfer sample at atmospheric pressure from a syringe to a Sample Loop. The loop is then connected via a change in valve configuration, to the high-pressure mobile phase stream, which carries the sample onto the column. There are two methods of loading the sample:

Complete-filling –where the loop size chosen has the desired injection volume and is totally filled with sample

Partial-filling –where the loop chosen is at least twice the required sample volume and is only partially filled

Dual mode injectors allow both complete and partial filling, single mode injectors allow only complete-filling. These two techniques differ in accuracy, precision and the amount of sample required and will be discussed further in a later topic. Steps in a manual injection process. Step 1: The sample is introduced using a syringe into the sample loop via a port on the injection valve. Depending upon the injection type –the loop will either partially or completely filled.


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Step 2: Once the sample is loaded the valve is turned to the ‘inject’ position. The rotor seal channels move and connect different ports on the stator. The pump is now able to push mobile phase through the sample loop, transferring the sample onto the column.

Step 3: Once the injection has been made the valve is returned to the ‘load’ position. This enables loop filling for the next injection. Some workers prefer to leave in the inject position until the next injection is required to completely flush the loop and reduce ‘carry-over’.

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Manual sample injectors possess different filling characteristics to automated sample injectors. In a manual sample injector the mobile phase that flows through the sample loop in the inject position is trapped when the rotor seal is returned to the load position. As the next sample is loaded, pushing mobile phase ahead of it, the front of the sample becomes diluted. This happens because the fluid has a parabolic velocity profile between the tube walls. At the centre of the tube the velocity is about twice the average, and at the wall the velocity is zero. This can result in wider that expected peaks. The effect is less noticeable in Gradient HPLC as the analyte tends to ‘focus’ as a tighter band at the head of the HPLC column under low solvent strength conditions at the beginning of the gradient.

Manual Injection Complete and Partial Loop Filling Various options exist for the injection of a given volume of sample solution into the flow of mobile phase –most methods use a loop injector and a pre-determined filling regime. Traditional loop injectors can be filled in one of the two ways outlined below: Complete Loop Filling –As the loop contains mobile phase, the sample solution introduced will necessarily mix and become diluted with the resident mobile phase whilst it is in the process of displacing it. Therefore, in order for the loop to be filled with homogeneous sample it should be overfilled between 2 and 5 times with the sample solution thereby eliminating any possible dilution effects. A 20 μL injection loop should be filled with between 40 and 100 μL of sample for example.

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Although the loop effectively controls the volume of sample injected, effectively making the amount of sample introduced from the syringe a non-critical parameter, it is recommended practice for maximum precision and linearity to overfill the loop with the same volume of sample solution each time i.e. 3x overfill ±10%, therefore for a 100μL loop this would equate to a sample volume of 270-330μL per injection. Reproducibility is essential in maintaining precision and accuracy; the absolute volume injected is of less importance. The figure summarises the dilution effeect on the reported peak area of various sample injections using a fixed injection loop volume of 20μL:

When less than10μL of sample is loaded there is a linear relationship between the sample volume placed in the loop and the amount of sample injected on to the column (as measured by peak area). When more than 40μL is used, then the injected volume is ±5% of the loop volume. However, in the 10 40μL injection volume range a distinct non-linear relationship can be clearly observed, and the reproducibility of the injection volume will be poor. After injection -10 loop volumes of mobile phase are required to completely displace the loaded sample from the loop. Therefore, it is recommended to leave the injector in the "inject" position to allow adequate time for the mobile phase to fully flush the loop, and to reduce sample ‘carry-over’ to a minimum. Partial Loop Filling -The partial loop filling technique is typically utilised when it is important to preserve valuable or limited sample, or if the correct sample loop is not available for the injection volume required. To obtain the best possible precision the volume of sample solution injected should be no greater than 50% of the injector loop capacity. A 100 μL loop should not be used for injections of over 50 μL for example. During the loading phase, the sample displaces mobile phase from the loop, and during this exchange process the front of the sample band becomes diluted. The consequence of this phenomenon is that the diluted sample occupies approximately 2μL of loop volume for every 1μL of sample loaded from the syringe. Therefore, by ensuring that less than 50% of the loop volume is used prevents the diluted front of the sample band from leaving the loop causing associated precision and irreproducibility errors.


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Autosamplers Autosamplers are widely used in analytical laboratories to increase sample throughput, improve injection precision and enable unattended operation –so reducing the labour costs associated with manual injection. Most autosamplers use six-port loop injection valves in order to deliver the sample plug to the analytical column. In modern autosamplers the rotor is driven by an electric motor, in older models, compressed air may be used. All autosamplers have the same basic components which include, the injection valve, a syringe or sampling needle, a loop of either fixed or adjustable volume, a metering pump to aspirate the sample from the vial and an injection port through which the sample is introduced into the loop. There are three main operating principles which are used in autosampler design:

1. Pull-to-fill 2. Push-to-fill 3. Integral-loop autosamplers

Each of these variations are implicitly different and have different performance characteristics.

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Whilst manufacturers have developed different autosampler operating principles and strategies, all possess four essential components that allow the effective mechanical automation of the manual injection system: 1. Samples are held in standard sized vials. Each sample vial is sealed with a septum, that is either an integral part of the cap or held securely in place by the cap, to prevent selective evaporation of the sample solvent causing associated concentration changes.

2. Sample vials are contained within trays that permit either their serial or random injection order.

Well plates and mixed autosampler trays for smaller injection volumes

Such trays can be thermostat controlled, helping to prevent degradation of thermally labile samples. It is possible to sample from vials or from well-plates or a combination of both. 3. An injection needle is employed to penetrate the septum, and draw the specified sample volume for injection using a highly accurate metering device or small analytical pump head. Depending on the autosampler employed such a needle may either be moveable or fixed.


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4. An injection switching valve that allows the introduction of the sample into the loop prior to injection. Such automated sample injection valves are operated either by pneumatic or electric actuators.

Pull to Fill Auto Samplers The Pull to Fill autosampler design draws sample solvent into the injection loop under syringe suction. A syringe or metering pump device is attached to the sample port of the injector. The needle is inserted into a sample vial and the syringe is drawn back to fill the loop with the desired volume. The syringe drive or pump stepper motor are accurately calibrated to ensure a reproducible volume of sample is withdrawn from the sample vial each time. During the injection phase the valve configuration allows the mobile phase to flow from the pump directly to the analytical column –effectively by-passing the loop. The rotation of the valve results in the injection of the contents of the sample loop, which are swept onto the column by the mobile phase flow.


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This autosampler design is quite simple, often with a needle that moves in a vertical plane with either a rotating sample tray or articulated arm movement selecting the desired sample vial. As the main components of the autosampler are not directly flushed by mobile phase during the injection step –it is necessary to flush the sample syringe and needle with a wash solvent between injections in order to reduce carryover. The wash solvent is dispensed to waste. Due to its simplicity and reliability this design was very popular, but is now much less popular due to the other two autosampler designs. Step 1: This is the ‘inject’ configuration. Mobile phase flows from the pump, through the sample loop and into the column. The valve orientation effectively isolates all other autosampler components from the eluent hydraulic path.

Step 2 –Load: The injection valve rotates to direct the eluent from the pump straight onto the analytical column. The syringe or metering pump is now used to PULL an exact volume of sample from the vial and into the sample loop –this can be used in either the full or partial loop mode.

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Advantages

Simple mechanical design which often translates to greater mechanical reliability

Relatively inexpensive and with very low maintenance costs Disadvantages

Many have no independent flushing procedure. Wash Vials are often placed between each sample on the carousel. This limits sample throughput

Suffers from the fact that there is excessive loss of sample during the injection sequence. As presented, sample solvent must necessarily fill both the needle and its connective tubing before reaching the sample loop, and consequently as much as ~ 10 - 100μL of sample is wasted per injection

Mechanical simplicity of the pull-to-fill designs often mean that the samples are placed in a tray that indexes one position at a time, so the vials must be loaded in the injection order. It may also require that the same number of injections be performed for each vial

Step 3 –Injection: The injection valve rotates again to direct the mobile phase through the loop –effectively displacing the analyte band onto the analyte band onto the analytical column. The rest of the autosampler is once again isolated from the hydraulic pathway.

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Step 4 –Wash: The syringe and needle flow path through the valve are rinsed with a wash solvent to reduce the amount of sample ’carry-over’ from one injection to the next. This operation is carried out multiple times with a solvent in which the analyte and sample components are highly soluble. The wash solvent is discarded to waste.

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Push to Fill Auto Samplers A more common autosampler design uses the ‘push to fill’ technique. This technique is very similar to manual injection. The syringe moves to the sample vial, draws the desired volume, moves to the injection port, and delivers the sample to the sample loop. Typically users need much less excess sample with this design than the pull-to-fill design.

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Syringe filling and dispensing is under the control of a stepper motor, which can provide very precise and accurate sample delivery. As a result errors of less than 0.5% are common in both filled and partial loop modes. Usually push to fill autosamplers add features to the basic pull to fill designs, such as random vial access, more effective flushing and programmable sample volumes. The tray layout may rotate or use an articulated arm movement that moves to find the desired vial from a rack of vials or a well plate. The valve can accommodate various volumes of sample loops although typical volumes range from 10μL to 500μL. Step 1: This is the ‘inject’ configuration. As with the Pull to Fill configuration the mobile phase flows from the pump through the valve directly to the analytical column, bypassing the other autosampler components.

Step 2 –Load: The needle moves to the sample vial and the metering pump is used to aspirate the sample into the holding loop. The injection valve switches and now the mobile phase into flows directly from the pump to the analytical column. This allows the

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metering device to PUSH the holding loop contents into the sample loop. The mobile phase which is displaced by the sample is directed to waste via the valve waste port.

Step 3 –Injection: The valve switches and the mobile phase is now directed through the sample loop to push the sample onto the analytical column. Once again the other components of the autosampler are isolated from the main hydraulic path of the eluent.

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Step 4 –Wash: A wash solvent may be used at this point to flush any remaining sample components out of the system. he analyte and other sample components should be highly soluble in the wash solvent. ith good washing and rinsing regimes it should be possible to attain carry over of very much less than 0.1%.

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Advantages

Usually allows random access to vials, a variable number of injections and a variable injection volume for each vial. Programming is simplest if the vials are injected in order, but the flexibility of random access can be a useful feature, particularly if one or more samples or standards must be injected at intervals during the run sequence

Generally provides the most flexibility in terms of injection volume. Small volumes of as little as 1-2μL can be injected if the syringe mechanism is driven precisely. By fitting a large loop on the injector, it is possible to inject a wide range of sample volumes and it is often possible to select the desired sample volume from the instrument control software

The low-pressure seal is rarely a problem and it can be easily adjusted by tightening a fitting on most models (This is in contrast to the high pressure seal on Integral Loop Autosamplers)

Disadvantages

Sample wastage is much lower than the ‘Pull to Fill’ Autosampler but is greater than is seen with most ‘Integral Loop’ Autosamplers

Mechanically more complex than the simple Pull to Fill Design’. Faults can be harder to diagnose

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Integral Loop Auto Samplers In recent years, the integral loop autosampler has become popular. The strong point of this design is that no sample is wasted and this can be very important for trace analysis when sample volume is limited. Because the sample is contained completely within the swept portion of the loop, the entire volume of loaded sample is injected. Additionally, continual flushing of the injection switching valve and loop following injection helps eliminate sample carryover effects. The maximum sample volume that can be injected is a function of the integral loop size, typically 100 μL, there is no practical lower limit to the injection volume. Manufacturers typically offer a multidraw option to compensate for this injection volume limitation. When connected the multidraw option allows for injection volumes of 100 μL to be "pooled", up to a typical maximum of around 1,500 μL, thereby increasing the volume injected and enabling trace sample analysis. The weakest part of this autosampler design is the high-pressure seal, which with the repeated needle insertions will eventually wear and leak, requiring replacement. Step 1: This is the inject configuration. The eluent flows from the pump, through the valve and into the syringe or metering device, through the needle, the high pressure seal and then on towards the analytical column. This configuration ensures that during analysis, all major components of the autosampler are flushed to reduce potential carryover.

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High Pressure Needle Seat: The sample first starts to mix with the mobile phase in the capillary that connects the high pressure needle seat to the rotor seal. Sample or mobile phase buffer precipitation may occur here if the polarity of the sample diluent and mobile phase differ greatly. Increased back pressure during the injection phase can indicate problems with precipitation in this area. Step 2 –Load: The injection valve turns to the ‘load’ position, the eluent flows directly from the pump to the analytical column. The autosampler components are now isolated from the main hydraulic flow path. The sample is moved into position, or the sampling needle arm moves to the sample depending upon design. The sample is aspirated into the integral loop using the metering pump or syringe device.

Step 3 –Injection: The valve switches to connect the autosampler components into the main hydraulic path. The eluent displaces the sample plug from the loop and onto the analytical column. The eluent flow path remains in this configuration to ensure good flushing of the autosampler. In some instances where the extra column volume need to be minimised.

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Advantages

Usually allows random access to vials, a variable number of injections and a variable injection volume for each vial. Programming is simplest if the vials are injected in order, but the flexibility of random access can be a useful feature, particularly if one or more samples or standards must be injected at intervals during the run sequence

Because the entire sample is contained in the swept needle-loop tubing, the integral loop autosampler wastes little or no sample. If microvials or vial inserts are used, almost all of the sample in the vial can be drawn into the sample loop and then injected. This feature is highly useful when trace analysis is being performed, as sample volume is often limited

The entire injection system is flushed with mobile phase for the vast majority of the run time. This helps to minimise carry over and contamination

Disadvantages

Allows programmed injection volumes but it can be difficult to change the loop so the upper end of the injection volume range is more restricted than it is for the push to fill models, although large volume adaptor kits are becoming more readily available for the integral loop design systems

The high-pressure seal is one of the weak points of the integral loop samplers and care should be taken to ensure that the seal is in good condition

Mechanically the most complex autosampler design. Faults can be more compled to diagnose

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Autosampler Contamination The sample first makes contact with the HPLC instrumentation in the Autosampler. Whist manufactures produce parts from the most inert materials possible (whilst considering cost implications), and to the best standards of design and engineering - the autosampler is still subject to contamination. Contamination is often presented in the form of sample carryover as evidenced when injection of a solvent blank produces a mini-version of the previous sample’s chromatogram. Most carryover occurs in the rotor seal via sample adsorption. Extra peaks / Ghost peaks that are sharp are often due to sample contamination rather than system contamination. The appearance of broad, less efficient peaks within otherwise reasonable chromatograms may indicate the elution of highly retained species from previous injections. Carry Over If a ‘mini’ version of the previous chromatogram is seen when injecting blank solvent, this is often called ‘carry over’. This maybe due to autosampler contamination –i.e. within the autosampler hydraulic path there is adsorption of the sample which is introduced into the column with the next injection plug. Autosamplers can become contaminated in several ways, usually due to a poorly swept component within the system, a component which is adsorptive towards analyte or sample components or due to poor needle rinsing.

System Contamination Extra peaks (often called ghost peaks) within the chromatogram that are broad in comparison to peaks around them indicate system contamination. This phenomenon is rarely due to autosampler contamination however and often indicates the very late elution of compounds that are strongly adsorbed within the analytical column. The peaks are broad due to the larger amount of diffusion that they


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Sample Contamination Extra peaks (often called ghost peaks) within the chromatogram that are approximately the same width in comparison to peaks around them indicate possible sample or autosampler contamination.

Rotor Seal Contamination: Sample carryover, in combination with poor peak area and height precision, indicates a worn rotor seal. The rotor seal should be evaluated for signs of wear, especially a deepening at the ends of the etched cross port grooves which can become sites of localised sample retention. These are then washed clean on the subsequent injection producing a mini-chromatogram or “ghosting” peaks.


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Sample carryover or ghosting peaks may also be the result of adsorbed material from the previous injection being desorbed into the new sample solution. Such sample adsorption can occur at various points through the injector system, and can be checked by flushing with 10 repeats of a solvent flush which are each 5 times the loop volume, followed by a re-injection of the blank. If no peaks are observed then either optimise the flushing routine or investigate the possible modes of chemical adsorption of the sample component(s) onto the injector component surfaces and correct as necessary. Sample Piston: The sampling piston in integral loop auto samplers should never come into contact with the sample provided the injection volume does not exceed the sample loop volume. Sample loop: It is uncommon for the sample loop to become contaminated. In ‘Integral Loop’ and ‘Push to Fill’ autosamplers the inside of the loop is constantly flushed with mobile phase. When using ‘Push to Fill’ autosamplers designs –‘flush’ injections can be used between sample injection to wash out the loop to prevent sample siphoning back into the loop. Therefore flush volumes should be chosen to be large enough to flush not only the loop but all of the tubing leading to the waste reservoir. Needle Seat: Both high and low-pressure needle ports at risk of contamination and blockage. The needle seat can be flushed directly by removing it from the autosampler and connecting directly to the outlet from the pump. In some autosampler designs the needle port will be flushed by the eluent during analysis. Eventually most port/seal designs will wear and leak, these then need to be replaced. Lifetime of the seal will depend on the number of injections and the alignment of the seal with the injection syringe which should be regularly checked. Autosampler Needle: The needle is subject to possible sample contamination. As a result needle washes are often used. This is usually accomplished using a wash solution held in a reservoir into which the needle is dipped and the wash solution drawn through the various system components where contamination might occur. The wash solvent is chosen on the basis of it being able to solubilise the potential contaminants. In ‘Integral Loop’ autosamplers the inside of the needle is constantly flushed with mobile phase, as a result, only outside of the needle needs to be cleaned between injections. Sample needle blockages are the most frequently occurring autosampler problem. Judicious sample preparation will help minimise the number of problems created by particulate material, with either filtering or centrifugation of the sample solution prior to injection helping to minimise the problem. A recommended cleaning regime for a blocked needle is as follows:

1. Soak the needle in an appropriate solvent i.e. isopropanol. Do not sonicate the needle if the component has been welded, as the possibility exists that it may fracture or break at the joint.

2. Attempt to dislodge the obstruction with a reaming wire. 3. Connect the needle to the pump head with a length of capillary and flush with

progressively higher flow rates to “blow out” the blockage

theory of hplc - chromacademy · instrumentation of hplc ... to describe the individual components...

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