Table A-1 Operational Specifications

Item Specification
Number of eluents One to four
Modes of operation Gradient, isocratic, and flow
programming
Operating flow range 0.00 to 45 mL/min (225 μ L heads),
in 0.01-mL increments.0.00 to 20
mL/min (100 μ L heads), in 0.01-
mL increments.
Composition range 0 to 100% programmable in 1%
increments for each of four
reservoirs: A, B, C and D. Total
composition must sum to 100%.

Compositional accuracy Better than 1.0%, independent of

Automatic eluent sparging
Pressure maxima 6000 psi
Programmable pressure
pressure.
Standard, . helium gas, input gas
pressure range 50 to 150 psi (3.5
to 10.5 kg/cm2). Helium flow rate
range 0 to 100 mL/min,
programmable in 1 mL/min
increments.
. (420 kg/cm2) at 10 mL/min.1000
psi (70 kg/cm2) at 45 mL/min.
Limits Lower: 0 to 5950 psi (0 to
416.5 kg/cm2)
Upper: 51 to 6000 psi (3.6 to 420
kg/cm2

This installment is the second of a series of four white papers on high-performance liquid
chromatography (HPLC) modules focusing on pumps, autosamplers, ultraviolet (UV)
detectors, and chromatography data systems, respectively. This installment provides a
technical overview of HPLC autosampler design, historical perspectives, and recent trends.
Recommendations for selection of autosamplers and guidelines for operating and
troubleshooting are provided.
The autosampler, an automated sample injector, introduces a precise aliquot of a sample solution
from a sample container to the high performance liquid chromatography (HPLC) column under
high-pressure flow conditions. It is a sophisticated automation device, capable of great precision
and long-term reliability. The historical evolution of manual injectors and auto-samplers have
been reviewed in many textbooks (1–4) and journal articles (5). Their performance, pressure
ratings, and reliability have increased dramatically in the last two decades to accommodate
demands from the use of narrower columns in ultrahigh-pressure liquid chromatography
(UHPLC) (6–9). In this article, an overview of HPLC autosamplers is provided by describing
their requirements, historical perspectives, modern design and trends, operating principles,
sampling configurations, and common operating, maintenance, and troubleshooting procedures.
The focus here is on analytical-scale applications, which require high-precision analysis in
regulated laboratories.

Glossary of Key Terms and Definitions

 Autosampler: A device that automates the introduction of an aliquot of sample solution

to the HPLC column. An HPLC autosampler typically comprises a sample storage
compartment with an injector, consisting of a valve, a sample dosing or metering device,
and a moving sampling needle.
 Injection Valve: A device such as a rotary valve that allows the introduction of a sample
solution in the HPLC column under high-pressure and flow conditions. An injection
valve can be a standalone manual valve, or part of an autosampler. It typically consists of
a needle port, a rotor and stator combination, and an exchangeable sample loop.
 Pulled-loop, pushed-loop, or split-loop design: These are the three common modes of
sample introduction designs. A split-loop design includes the sampling needle as part of
the sample loop, and aspires an exact sample aliquot to be introduced. A pulled-loop
design aspires the sample by vacuum from an external sampling syringe into the loop. A
pushed-loop design fills a loop with pressure.
 Full-loop or partial-loop mode: Two modes of injection are used with pulled- or
pushed-loop autosamplers. In a full-loop (or fixed-loop) mode, the entire sample loop is
filled with the sample solution. In a partial-loop or variable-loop mode, only a fraction of
the loop (10%–50%) is filled. The full-loop mode is more precise, particularly for small-
volume injections, but requires more sample solution to overfill the loop (three times the
sample loop volume to ensure that the entire loop is filled with the sample solution). The
partial-loop mode offers more flexibility than the full-loop mode by its ability to inject
different sample volumes without a physical change of the sample loop.
 Sampling syringe or metering device: A precise volume dosing unit required for
sample aspiration. Two types are commonly used. One is a flow-through metering
device, which is a part of the high-pressure flow path of a split-loop autosampler; the
other is a low-pressure dead-end sampling syringe.
 Sample container: Typically, a 2-mL glass vial containing the sample dissolved in a
diluent (final sample solution). Vials are placed in an autosampler-compatible vial holder,
typically rectangular vial trays (vial racks), or vial carousels. Other configurations are
microplates, commonly used in high-throughput screening (HTS) applications.
  Sampling needle: Typically, a 22-gauge blunt-tip needle of a manual sample syringe
connected to the end of a movable stainless steel or polyether ether ketone (PEEK)
sampling capillary, typically used with pushed-loop type. Split-loop autosamplers use
highly specific sampling needles that match the needle-port, and must be capable of
operating at high pressure.
 Sample compartment: An enclosed compartment for storing sample containers, which
is commonly thermostated. An optional device (plate changer) can be used to transport
additional sample trays or microplates, and increase sample capacity.
 Needle wash port: A needle port connected to waste for parking the sampling needle and
rinsing the outside of the sampling needle with a solvent that is supplied by a peristaltic
pump (or by the sampling syringe flushing through the inside and outside of the needle).
The needle wash is used to reduce carryover from the previous sample, which adheres to
the outside of the needle.
 Needle seat/port: For pushed-loop autosamplers, an orifice through which the sampling
needle can fill the sample loop; for split-loop autosamplers, a counterpart port for the
sample needle that must seal to the maximum system pressure level. This port is not
required for pulled-loop autosamplers.
 Carryover: The amount of the main component from the prior injected sample, which is
observed in a subsequent injection of the sample diluent (blank) without analyte.
Carryover performance is highly analyte-dependent, and is particularly problematic for
very basic compounds and proteins.
 Injection volume precision: Precision reflects the variation (scatter) of the delivered
sample volumes during repetitive injections as a relative standard deviation (RSD) of
peak area or peak height.
 Injection volume accuracy: Accuracy describes the deviation or systematic error of the
absolute injection volume from the intended volume. The volume accuracy requirement
is less relevant in practice, since method calibration (standardization with a reference
compound) is performed in quantitative HPLC analysis.
 Injection cycle time: The time required to accomplish a sample injection that includes
device movements to bring the sampling needle to the sample, sample volume aspiration,
and needle wash or loop cleaning. The injection cycle can be reduced by overlapping
with the ongoing separation cycle time (if allowed by the instrument and control
software).
 Pressure transducer: The pressure sensor in the autosampler flow path used in some
autosamplers to monitor the injection sequence. The pressure sensor can be used to
optimize the injection routine timing, or warn the customer in case of any irregular
behavior.

Solvents used in the autosampler include:

 Wash solvent: the rinsing liquid(s) used to clean the sampling needle and loop to reduce
carryover.
 Carrier solvent: the liquid used to aspire the sample into the sampling needle, needle
capillary, and sample loop, typically in direct contact with the sample solvent. The
mobile phase can also be used as a flush or carrier solvent in some designs (the flow-
through metering device in split-loop samplers).
  Diluent: the solvent for diluting the sample in the final sample solution, which should be
weaker than the initial mobile phase, to avoid potential peak distortions. Note that the
sample injection volume is proportional to the column void volume (3).

Analytical Column
HPLC columns are usually stainless steel columns with end fittings to retain packing material. These are
most commonly packed with micro-particulate silica. The packing material is the stationary phase.
Gradient Elution
A predetermined change in mobile phase composition during separation e.g. 40% methanol/60% water
increasing in a linear step to 80% methanol/20% water during the run.
Isocratic Elution
When there is constant solvent composition throughout chromatographic separation.
HPLC Analytical System
An analytical system consists of solvent reservoir(s), high/low pressure pump(s), an injection system, an
analytical column, a UV/Vis absorbance and/or fluorescence based detector, data acquisition software and
where required a column oven, sample cooler and/or a gradient controller.
High Performance Liquid Chromatography (HPLC)
This technique can be used for almost any sample that dissolves in a solvent. By choosing an appropriate
column and mobile phase, desired separations can be achieved. The technique involves separation due to
differences in the equilibrium distribution of sample components between two different phases. One of
these phases is a solid stationary phase (the column), while the other is a moving liquid (the mobile
phase). The samples migrate through the chromatography system only when they are in the mobile phase.
Separation results from different velocities of migration as a consequence of differences in equilibrium
distributions.

 Mobile PhaseMobile
 Phases used in HPLC may be water, organic solvents or buffers either on their own or mixed
with one another. This acts as a carrier for the sample in the HPLC system.
 Normal Phase HPLC
 This mode of HPLC is used for separating compounds containing polar functional groups.
Separation is due to differences in the partition coefficients of solutes between the relatively polar
stationary phase and the relatively non-polar mobile phase.
 Reverse Phase HPLC
 This mode of HPLC is used for separating non-polar and moderately polar compounds.
Separation is due to differences in the partition coefficients of solutes between the relatively non-
polar stationary phase and the relatively polar mobile phase.

NO.
ADMS Atmospheric Dispersion Modelling System
APHA American Public Health Association
BOD Biological Oxygen Demand
CCS Carbon Capture and Storage
COD Chemical Oxygen Demand
DPSIR Driving force Pressure State Impact Response
EMIN EnvironmentalMonitoring and Indicators Network
EPD Environment Protection Department
EP&CCD Environment Protection and Climate Change
Department.
ICP Inductively Coupled Plasma
 IETT Institute of Environmental Technology and Training .
LIMS Laboratory Information Management System.
LIR Lab Inspection Report
MEAs Multilateral Environmental Agreements
NTU Nephelometric Turbidity
PCEI Provincial Core set of Environmental Indicator .
POPs Persistent Organic Pollutants
PVC Polyvinyl Chloride
SoE State of Environment
SoER State of Environment Report .
TDS Total Dissolved Solids.
TFE Tetrafluoroethylene
TSS Total Suspended Solids.
Pak-EPA Pakistan Environmental Protection Agency.
USEPA United States Environment Protection Agency.

Procedure for Changing Mobile Phase System When changing from Reverse
Phase to Normal Phase operation Wash the system with methanol: water 50:50 or wash as
recommended in the method for 30 minutes. Remove the analytical column. Insert a suitable connector
in place of the column. Draw through methanol and wash with 100% methanol for 30 minutes. Draw
through dichloromethane: methanol, 50:50 and wash for 30 minutes. Draw through the Normal Phase
eluant and wash for 30 minutes. Insert normal phase analytical column. Equilibrate with eluting solvent
for a period of 30 minutes, or until a stable baseline is obtained. Note that if changing a suitable column
(e.g. NH2) from reverse phase to normal phase, then leave column in place during change over.

When changing from Normal Phase to Reverse Phase operation

Wash the system with recommended wash solvent for 30 minutes. Remove the analytical column. Insert
a suitable connector in place of the column. Draw through dichloromethane: Methanol 50:50 and wash
for 30 minutes. Draw through and wash with 100% methanol for 30 minutes. Draw through and wash
with methanol: water, 50:50 for 30 minutes. Draw through and wash with Reverse Phase eluant and
wash for 30 minutes. Insert reverse phase analytical column. Equilibrate with eluting solvent for a period
of 30 minutes or until a stable base-line is obtained. Note that if changing a suitable column (e.g., NH2)
from Normal Phase to Reverse Phase, then leave column in place during change over.

During equilibration 1 Check for any system leaks2 Ensure that the pressure of the pump is a
steady value; if this is not the case then flush the pump 3 If the baseline on the chromatogram does not
stabilise and exhibits erratic behaviour (particularly shooting off scale), then there is likely to be air in the
detector cell. This should be primed by gently pushing mobile phase through the cell using a syringe 4 If
the baseline exhibits regular stepping patterns, either peaks or troughs, this means that the detector lamp
is deteriorating and requires changing 5 If there is an absolutely straight baseline ensure that all electric
connectors are in place and that the detector lamp is lit 6 If the expected profile is not exhibited during a
blank gradient run. Ensure that the correct reservoirs are being used Check that the programme has been
set correctly and the correct wavelength is being used Ensure the pump(s) and the mixing valve are in
working order Ensure that the correct absorbance wavelength and/or the correct emission and excitation
wavelength are selected for UV/Vis absorbance and/or fluorescence detection respectively 7 Prepare
system suitability/standards/control/samples as directed in the appropriate analytical method

Starting a run 1 Set up the sample run using the appropriate sample set method 2 Check that the HPLC
conditions are stable and ready for testing to commence 3 If the instrument is ready, start the run
After Use 1 Calculate the results from the collected data as directed in the appropriate analytical method 2 After
all work is complete, flush the HPLC system with an appropriate wash solvent (refer to Analytical Method) 3 If the
system is not going to be used the next day, then switch off the detector lamp, column heater and pump flow
No. Name of Industry Contact

ANKLESARIA HOSPITAL https://www.indeed.com.pk/Hospital-jobs-in-Karachi 02132729719

BAQAI HOSPITAL https://baqai.edu.pk/Jobs.php. 02136618396
SOUTH CITY HOSPITAL (PVT)
LTD.
http://southcityhospital.org/index.php/careers. 02135862301

04236817860
https://www.indeed.com.pk/Shalamar-Hospital-,-Lahore-jobs-in-
SHALAMAR HOSPITAL
Pakistan.

EXCEL LABS (PVT) LTD https://www.rozee.pk/company/excel-labs. 0512829869

ASIA LAB DIAGNOSTIC http://www.jobz.pk/asia-diagnostic-centre-lab-technicians-

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02132729043
https://www.indeed.com/q-Medical-Lab-Technician-
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Central-Lab-jobs.html.

CITY LAB X- RAY AND http://www.jobz.pk/citi-lab-lahore-jobs-2019-for-x-ray-

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02136315886
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http://www.pakistanbusinessjournal.com/b2b-
Dr. MEHDI A. MANJI 02134555952
directory/apply_for_job_dr-mehdi-a-manji_9715.html.
AL-NASAR LAB https://www.rozee.pk/company/al-nasar-lab. 04235163924
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QURESHI PATHOLOGY
LABORATORIES (PVT) LTD
https://www.rozee.pk/company/wilshire-laboratories-pvt-ltd. 024235852473

VITAL MARK LABORATORIES

(PVT) LTD
http://www.jobz.pk/company/vital-mark-laboratories/. 04237595708

DOCTORS LAB https://www.indeed.com/q-Doctors-Lab-jobs.html. 0514413975

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https://www.rozee.pk/company/dr-essa-laboratory-diagnostic-centre. 02136626125

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