HPLC autosampler glossary: 72 terms explained

This HPLC autosampler glossary defines 72 terms that turn up in autosampler manuals, datasheets, method documents and integration specs, from backflush to virtual COM port. Each entry gives a short definition, a typical value or unit where one helps, and, where we have one, a link to the post that covers the topic in depth. Terms are in alphabetical order; synonyms point to the main entry.

Key points

• Most autosampler specifications come down to four numbers: injection precision (RSD, %), carryover (%), injection volume range (µL) and pressure rating (bar or psi).
• There are two main injection architectures, the flow-through needle (also called split-loop or integrated loop) and the fixed loop (pulled or pushed loop). Several terms below only apply to one of them.
• Pressure units: 1 bar = 14.5038 psi, 1 MPa = 10 bar, so a 5000 psi valve is a 345 bar valve.
• Regulatory definitions of carry-over, LLOQ and precision used here come from the ICH M10 guideline; system suitability limits come from pharmacopoeia chapters such as USP <621> and Ph. Eur. 2.2.46.

A

Air gap

A small volume of air drawn between two liquids in the needle or syringe line so that they do not mix, for example between a wash solvent and the sample. Whether a given autosampler uses air gaps depends on its fluidic design and injection program.

Aliquot

A measured portion withdrawn from a sample for analysis or transfer, for example 10 µL taken from a 1.5 mL vial or 50 µL taken from a reactor.

Aspiration

Drawing liquid into the needle, loop or syringe by moving the piston of the metering device. The volume and speed of aspiration are the two main settings; see draw speed.

Autosampler

An instrument that takes samples from vials or well plates and introduces a defined volume into the HPLC flow path without manual handling. Vendors also call it an automatic injector, sample manager or sampler. The injection principles differ more between designs than the outside suggests.

Automated dilution

An injector program step in which the autosampler draws sample and diluent, dispenses both into an empty vial and mixes them before injection. The LCGC review of autosampler design lists dilution and pre-column derivatisation as typical extra liquid handling steps, while noting they are used infrequently (LCGC, 2019).

B

Backflush injection

An injection in which the mobile phase pushes the loop contents to the column in the opposite direction to the one in which the loop was filled. The sample sits at the loop inlet, so it leaves by the shortest path instead of travelling through the whole loop, which matters most for partial-loop injections. See full-loop vs partial-loop injection.

Backpressure

The pressure the pump must generate to push mobile phase through the column and the rest of the system. It scales with flow rate, viscosity and column length, and with the inverse square of particle size: a 4.6 × 150 mm, 5 µm column at 1 mL/min gives roughly 45-80 bar at 25 °C, depending on the mobile phase (worked estimate).

Bar

Unit of pressure: 1 bar = 100 kPa = 0.1 MPa = 14.5038 psi. European and Japanese vendors usually rate pumps, valves and columns in bar or MPa.

Blank injection

An injection of a solution that contains no analyte (pure solvent, or matrix without analyte). Blanks placed after high-concentration samples are the standard way to measure carryover.

C

Carryover

Signal from a previous sample that appears in a later injection, usually expressed as a percentage of the preceding peak area. ICH M10 defines carry-over as "an alteration of a measured concentration due to residual analyte from a preceding sample that remains in the analytical instrument" and, for bioanalytical chromatography, limits the blank after the highest calibrator to 20% of the LLOQ response (ICH M10). How to measure and reduce it: autosampler carryover.

CDS (chromatography data system)

Software that controls the LC modules, acquires detector signals, integrates peaks, calculates results and keeps an audit trail. Examples are Waters Empower, Thermo Scientific Chromeleon and Agilent OpenLab; each needs a driver for every instrument it controls.

Contact closure

A simple electrical signal between instruments, typically a relay or open-collector output that is either open or closed. In LC it is used for events such as "injection now" (inject start) or "ready", so that a pump, detector or CDS from another vendor can start its run at the moment of injection. It carries timing, not data.

Crimp cap

An aluminium cap with a septum that is pressed onto the vial neck with a hand or electric crimper. It gives a consistent, tight seal and suits volatile samples, at the cost of an extra tool and step. See vials, caps and septa.

Cross-port leakage

Leakage between neighbouring ports of a rotary valve across a scratched or worn rotor seal or stator face, so that liquid passes where the valve position says it should not. Symptoms include drifting peak areas, ghost peaks and carryover; see the 6-port injection valve.

Cycle time

The time from the start of one injection until the autosampler is ready for the next, including needle moves, aspiration, washing and valve switching. The 2019 LCGC review cites a design with injection cycles "down to 8 s".

D

Dead volume

A volume in the flow path that the flow does not sweep efficiently, such as a gap at a badly seated fitting or a dead-end cavity. It holds sample and releases it slowly, which causes peak tailing and carryover. Some authors use the term loosely for extra-column volume; "unswept volume" is the clearer name.

Delay volume

The volume a liquid must pass through before a change reaches the point of interest. In gradient LC it normally means the gradient delay volume; in reactor sampling it is the volume of the transfer line between the sampling point and the injector, which sets how old the injected sample is. See online reaction monitoring.

Derivatisation

A reaction of the analyte with a reagent to make it detectable or better separated, for example o-phthalaldehyde (OPA) with primary amino acids for fluorescence detection. Many autosamplers can mix sample and reagent in the needle, loop or a vial and wait a set time before injection (pre-column derivatisation).

Draw speed

The rate at which the metering device aspirates liquid, in µL/s or µL/min. Drawing too fast from a viscous or volatile sample can pull bubbles or leave the drawn volume short, so viscous samples need slower draw speeds and sometimes a wait time after drawing.

Dwell volume

See gradient delay volume.

E

Extra-column volume

All the volume between the injection point and the detector outside the column: loop or needle, connecting capillaries, fittings, heat exchanger and flow cell. It broadens peaks, which matters most with short, narrow columns and small particles, and is one reason UHPLC systems use narrow capillaries.

F

Fixed-loop autosampler

A design in which the sample is placed into a sample loop mounted on the injection valve, separate from the needle. The volume is set by the loop (full-loop injection) or by the metering syringe (partial-loop injection), and the needle and syringe stay at low pressure. The LCGC review calls the two variants pulled-loop and pushed-loop.

Flow-through needle

A design in which the needle, needle seat and metering device are part of the high-pressure flow path: after the needle draws the sample it returns to the seat, the valve switches, and mobile phase flows through the needle to the column. The LCGC review notes it is "often called integrated-loop, flow-through needle, or needle-in-loop design". All drawn sample is injected and the sample path is flushed by mobile phase during each run.

Fraction collection

Collecting a liquid stream (column eluate, reactor samples or diluted aliquots) into vials or wells in portions defined by time, volume or a detector signal. Autosamplers with a liquid-handling configuration can do simple fraction collection into vials.

Full-loop injection

An injection in which the sample loop is completely filled, so the injected volume equals the loop volume. Because of the parabolic flow profile the loop must be overfilled; the LCGC review recommends "a minimum of three times the sample loop volume". It is the most precise mode but uses the most sample; see full-loop vs partial-loop injection.

G

Gradient delay volume (dwell volume)

The volume between the point where the gradient solvents are mixed and the column inlet. It delays the gradient by V/F: a 1 mL delay volume at 1 mL/min means the column sees the programmed change 1 minute late. Differences in delay volume between systems are a common reason why gradient methods transfer badly; the autosampler's contribution is usually small compared with the pump's, according to the LCGC review.

I

Injection precision

How repeatable the injected amount is, measured as the RSD of peak areas from replicate injections of the same solution. The LCGC review gives "<0.5% RSD for regulatory analysis" as the key requirement. How to test it: autosampler qualification.

Injection valve

A rotary valve that switches the sample loop (or the needle path) into or out of the mobile phase stream. The most common type is the 6-port, 2-position valve with a load and an inject position; see the 6-port injection valve explained.

Injection volume

The volume of sample introduced into the column, typically 1-100 µL on analytical HPLC. It is set by the loop in full-loop mode and by the metering device in partial-loop and flow-through needle designs. How much a column tolerates: HPLC injection volume.

Injector program

A user-defined sequence of autosampler steps, such as draw, wait, mix, wash and inject, used for dilution, derivatisation or extra needle cleaning. Vendors use names such as injector program or pretreatment program.

Internal standard

A compound added at the same known concentration to every sample and standard. Quantifying by the ratio of analyte response to internal-standard response corrects for variation in injected volume and sample preparation.

L

LADS

The OPC UA Laboratory and Analytical Device Standard, published as OPC 30500 by a joint working group of the OPC Foundation, SPECTARIS and VDMA. It is a manufacturer-independent information model for lab instruments; Part 1, version 1.0.0, was published on 30 November 2023 (OPC Foundation). See SiLA 2, LADS and serial.

Leak sensor

A sensor under the valve, needle seat or syringe that detects liquid collecting in a leak tray or catch cup and stops the run or raises an error. It protects the electronics and flags a worn seal or loose fitting early.

Linearity (injection volume)

How well the peak area scales with programmed injection volume across the autosampler's range, tested by injecting several volumes from the same vial and fitting a straight line. It shows whether the metering device is accurate at small and large volumes.

LLOQ (lower limit of quantification)

ICH M10 defines it as "the lowest amount of an analyte in a sample that can be quantitatively determined using a method with predefined precision and accuracy". Carryover limits in bioanalysis are expressed relative to the LLOQ response.

Load position

The valve position in which the sample loop is out of the mobile phase stream and can be filled, while the pump flows directly to the column. The other position is the inject position.

M

Metering device

The precision syringe or piston unit that draws and dispenses sample by volume. In flow-through needle designs it sits in the high-pressure path; in fixed-loop designs it is a low-pressure syringe. Its stroke resolution and seal condition set the accuracy of small injections.

Microplate

See well plate.

MPa

Megapascal: 1 MPa = 10 bar = 145.04 psi. Shimadzu, among others, states pressure ratings in MPa, for example 130 MPa (1300 bar) for the Nexera X4 pump.

N

Needle depth

How far the needle enters the vial, usually set as an offset from the vial bottom. Raising it avoids drawing sediment; lowering it lets you sample small volumes from vial inserts.

Needle seat

The port into which the needle presses to form a seal. In flow-through needle designs the seat "must seal to the maximum system pressure level", while in pushed-loop designs it is a low-pressure port (LCGC). A worn or contaminated seat causes leaks, pressure errors and carryover; see autosampler troubleshooting.

Needle wash

Rinsing the needle between injections to reduce carryover: an external wash (dip in a wash vial or a flowing wash port) cleans the outside, and an internal rinse cleans the bore. The wash solvent should dissolve the analyte at least as well as the sample solvent.

O

OEM module

An autosampler sold as a component for another company's instrument, with mounting points, a low-voltage DC supply and a documented control protocol instead of a vendor CDS driver. What to check: OEM autosampler integration.

Overfill

The extra sample volume pushed through the loop in full-loop injection to replace the solvent completely; the LCGC review recommends at least three loop volumes. A 20 µL loop therefore consumes 60 µL or more per injection.

Overlapped injection

Preparing the next injection (drawing sample, washing the needle) while the current separation is still running, so the next run can start sooner. The LCGC review describes it as reducing the injection cycle "by overlapping with the ongoing separation cycle time".

P

Partial-loop injection

An injection in which only part of the loop is filled, with the volume set by the metering syringe; the LCGC review gives "10%-50%" of the loop as the usual fill. It wastes less sample and allows variable volumes but is less precise than full-loop injection.

PEEK

Polyether ether ketone, a metal-free polymer used for tubing, fittings, rotor seals and needle seats. It is chosen for chemical inertness and for metal-sensitive analytes; pressure ratings of PEEK parts are lower than steel, and some solvents and strong acids are not compatible, so check the supplier's chart.

Pre-slit septum

A septum with a cut through it so the needle penetrates easily. It reduces septum coring and the vacuum that builds up when liquid is drawn from a sealed vial, at the cost of slightly more evaporation. See vials, caps and septa.

Pressure drop at injection

A short dip in system pressure when the injection valve switches the loop, filled at atmospheric pressure, into the high-pressure stream: the liquid in the loop compresses and the pump has to make up the volume. The dip is larger with big loops and high pressures; see HPLC pressure ratings.

psi

Pound-force per square inch: 1 psi = 6.894757 kPa, so 1000 psi = 68.95 bar. US vendors and most fitting and tubing suppliers rate parts in psi.

Pulled-loop and pushed-loop

The two fixed-loop variants described in the LCGC review. In a pulled loop the syringe "directly draws the sample into the sample loop"; in a pushed loop the sample is first drawn into a capillary and then pushed into the loop through a low-pressure needle port.

R

Rack

The removable holder for vials or plates. Racks built to the SBS footprint fit plate handlers and robot grippers.

Rotor seal

The rotating polymer disc inside a rotary valve, with grooves that connect pairs of stator ports. It is the main wear part of an injection valve; vendors rate its life in switching cycles. See rotor seals and pressure.

RSD (relative standard deviation)

Standard deviation divided by the mean, times 100, also called the coefficient of variation (CV). ICH M10 expresses precision this way. Example: peak areas of 1000, 1004 and 996 have a mean of 1000 and a standard deviation of 4, so the RSD is 0.40%.

S

Sample loop

A length of tubing with a defined internal volume mounted on the injection valve. Its volume is V = π × (d/2)² × L: a 20 µL loop needs about 102 mm of 0.50 mm ID tubing, or about 407 mm of 0.25 mm ID tubing. See how to size a sample loop.

SBS footprint

The standard outer footprint of a microplate, 127.76 × 85.48 mm, defined in ANSI SLAS 1-2004 (originally an SBS standard) (SLAS). Vial racks and plates with this footprint fit plate handlers, hotels and robot grippers.

Screw cap

A threaded cap with a septum, closed by hand. It is quick and needs no tool; the seal depends on consistent tightening. See vials, caps and septa.

Seat backflush

Flushing the needle seat in the reverse direction to clean the sealing area between needle and seat. Agilent's carryover guide says "if the needle seat becomes contaminated, it must be back-flushed", and its multiwash option automates this with up to three solvents (Agilent).

Septum

The elastomer disc in a vial cap, usually silicone with a PTFE face towards the sample. The needle pierces it on every injection; cored or swollen septa cause blocked needles and contamination.

SiLA 2

An open standard for connecting lab devices and software, maintained by the SiLA consortium. According to the consortium it "runs over HTTP/2 and uses Protocol Buffers to serialize payload data", relying on the gRPC wire format (SiLA). See lab automation standards.

Split-loop

Another name for the flow-through needle design: the loop is split into two parts, with the needle between them, while the sample is drawn.

Stator

The fixed part of a rotary valve that carries the ports where tubing connects. The rotor seal presses against its polished face; a scratched stator causes cross-port leakage even with a new rotor seal.

System suitability

Tests run with each analytical sequence to show that the system performs as the method requires, for example replicate-injection RSD, resolution, tailing factor and signal-to-noise. Pharmacopoeia chapters such as USP <621> and Ph. Eur. 2.2.46 define them.

T

Transfer line

The tubing that carries liquid from a sampling point (reactor, flow cell, process stream) to the autosampler or injection valve. Its volume adds delay volume and must be flushed before each sample.

Tray thermostat

A Peltier-cooled (sometimes heated) sample compartment that keeps vials at a set temperature to slow degradation and evaporation. The LCGC review gives "around 4 °C" as the typical lower limit.

U

UHPLC

Ultra-high-performance liquid chromatography: LC with sub-2 µm particles on instruments built for pressures well above conventional HPLC. Older texts put the boundary at about 400 bar (6000 psi) (Dolan, LCGC); current UHPLC systems are rated 1000-1500 bar. Unit conversions and system classes: HPLC pressure ratings.

Unswept volume

See dead volume.

V

Valve switching time

The time the injection valve takes to move from one position to the other. During the move, ports are briefly blocked or connected in sequence, which causes a short pressure transient.

Vespel

A polyimide (a DuPont trade name) used for rotor seals and needle seats because it is hard and wear-resistant. It is not the best choice for strongly basic mobile phases or very basic analytes, which can adsorb on it and cause carryover; see rotor seal materials.

Vial

The sample container. The common autosampler vial holds 2 mL and measures 12 × 32 mm; it is closed with a screw, crimp or snap cap. See vials, caps and septa.

Vial insert

A small glass or polymer insert placed inside a standard vial to hold a few hundred microlitres or less, raising the liquid so the needle can reach it. Check that the needle depth setting suits the insert.

Virtual COM port

A serial port that the operating system creates when a USB device implements a serial class such as USB CDC ACM; on Windows 10 and 11 the in-box Usbser.sys driver loads for such devices without a custom driver (Microsoft). Software opens it like an RS-232 port (COM3 on Windows, /dev/ttyACM0 on Linux) and exchanges commands as text or bytes. See controlling serial instruments from Python.

W

Wash port

A station where the needle is rinsed between injections, either a static wash vial or a flowing port fed by a wash pump. A flowing port removes residue from the outside of the needle more reliably than a single dip.

Well plate

A plate with 96 or 384 wells (sometimes 24 or 48) in the SBS footprint, used for high-throughput work. The LCGC review notes microplates are "commonly used in high-throughput screening (HTS) applications".

Wetted materials

All materials that the sample or mobile phase touches: for example 316L stainless steel, PEEK, PTFE, FEP, Vespel and fused silica. They decide chemical compatibility, adsorption (and so carryover) and suitability for metal-sensitive analytes.