Instrumentation of HPLC

Autosamplers

Wherever you see this symbol, it is important to access the on-line course
as there is interactive material that cannot be fully shown in this reference
i manual.
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
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

© Crawford Scientific www.chromacademy.com 2

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

© Crawford Scientific www.chromacademy.com 3

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.

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

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.

© Crawford Scientific www.chromacademy.com 4

 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.

© Crawford Scientific www.chromacademy.com 5

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.

© Crawford Scientific www.chromacademy.com 6

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.

© Crawford Scientific www.chromacademy.com 7

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.

© Crawford Scientific www.chromacademy.com 8

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.

© Crawford Scientific www.chromacademy.com 9

 i

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’.

© Crawford Scientific www.chromacademy.com 10

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.

© Crawford Scientific www.chromacademy.com 11

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.
© Crawford Scientific www.chromacademy.com 12
 i

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.
© Crawford Scientific www.chromacademy.com 13
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.

© Crawford Scientific www.chromacademy.com 14

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.

© Crawford Scientific www.chromacademy.com 15

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.

© Crawford Scientific www.chromacademy.com 16

 i

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.

© Crawford Scientific www.chromacademy.com 17

 i

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.

© Crawford Scientific www.chromacademy.com 18

 i

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.

© Crawford Scientific www.chromacademy.com 19

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
© Crawford Scientific www.chromacademy.com 20
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.

© Crawford Scientific www.chromacademy.com 21

 i

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%.

© Crawford Scientific www.chromacademy.com 22

 i

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

© Crawford Scientific www.chromacademy.com 23

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.

© Crawford Scientific www.chromacademy.com 24

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.

© Crawford Scientific www.chromacademy.com 25

 i

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

© Crawford Scientific www.chromacademy.com 26

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

© Crawford Scientific www.chromacademy.com 27

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.

© Crawford Scientific www.chromacademy.com 28

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

© Crawford Scientific www.chromacademy.com 29