# How an HPLC autosampler works: injection principles explained

> How an HPLC autosampler works, step by step: needle, syringe, loop, valve and wash, plus fixed-loop, flow-through needle and split-loop designs compared.

Source: https://elutrolab.com/en/blog/how-an-hplc-autosampler-works
Published: 2026-10-13
Publisher: Elutrolab (https://elutrolab.com)

An HPLC autosampler uses a needle and a syringe (or metering pump) to draw a set volume of sample from a vial, places it in a sample loop, and then turns an injection valve so the pump's high-pressure flow sweeps the loop contents onto the column. Between injections it washes the needle and sample path so the next vial is not contaminated. The main designs differ in which parts sit in the high-pressure flow: fixed-loop autosamplers switch only the loop into the flow, while flow-through needle and split-loop designs keep the needle and loop in the flow path, trading extra gradient delay volume for zero sample loss.

## Key points

- Every autosampler has the same core parts: needle, syringe or metering device, injection valve, sample loop, wash station and a tray or rack.
- Fixed-loop designs fill the loop at low pressure, either completely (2-5 loop volumes of sample, best precision) or partially (no more than about half the loop, flexible volume).
- Flow-through needle and split-loop designs inject everything they draw, but the needle seat must seal at full system pressure and the needle and loop add to the gradient delay volume.
- The autosampler's share of the gradient delay volume ranges from tens to hundreds of microlitres. On a 2.1 mm column that can mean more than one column volume of extra isocratic hold.
- The best design depends on sample volume, required precision, column size and whether the autosampler must fit a compact or OEM system.

## What are the parts of an HPLC autosampler?

| Part | What it does | Typical limit or failure |
|---|---|---|
| Needle | Pierces the vial septum and draws sample | Bent tip, septum coring, residue on the outside (carryover) |
| Syringe or metering device | Sets the volume drawn in partial-fill modes | Worn plunger seal, air bubbles, larger relative error at small volumes |
| Injection valve | Switches the loop between the low-pressure fill path and the high-pressure column path | Worn rotor seal, cross-port leakage |
| Sample loop | Holds the sample until injection | Real volume differs from the nominal one |
| Needle seat or injection port | Connects the needle to the valve; in flow-through designs it seals at full system pressure | Leaks, blockage, carryover |
| Wash station | Rinses the needle outside (dip) and the sample path inside (rinse) | Wash solvent too weak to dissolve the sample |
| Tray or rack | Holds vials or plates at known positions | Wrong vial height or septum type |

The valve in almost every HPLC autosampler is a rotary 6-port, 2-position valve, explained port by port in our [guide to the 6-port injection valve](/en/blog/six-port-injection-valve). It has two jobs: route the pump flow away from the loop so the loop can be filled, then route it through the loop so the solvent carries the sample to the column ([IDEX fluidics FAQ](https://www.idex-hs.com/contact/contact-us/faqs/fluidics)).

## The injection cycle, step by step

This is the sequence for a fixed-loop autosampler; other designs vary steps 4 to 7.

1. **Standby.** The valve is in LOAD. The pump feeds the column directly; the loop is isolated and at low pressure.
2. **Needle to vial.** The needle moves to the programmed vial, pierces the septum and goes down to a set depth.
3. **Draw.** The syringe or metering device draws the programmed volume.
4. **Transfer to the loop.** Sample is pushed or pulled into the loop through the valve's sample port. Any excess goes to waste.
5. **Inject.** The valve turns to INJECT. Pump flow now runs through the loop and carries the sample onto the column. This moment is time zero of the chromatogram.
6. **Wash.** While the separation runs, the needle is rinsed outside and the sample path inside.
7. **Flush and reset.** The loop stays in the flow until the sample has been swept out, then the valve can return to LOAD for the next vial.

How long is long enough in step 7? IDEX recommends pumping at least ten sample volumes through the loop: if you load 20 µL into a 100 µL loop at 1 mL/min, stay in INJECT for at least 12 s ([IDEX injectors troubleshooting guide](https://www.idex-hs.com/resources/resources-detail/injectors-troubleshooting-guide), Appendix E). For 25 µL at 0.4 mL/min the same rule gives 37.5 s: flush time grows as flow drops.

## Fixed-loop autosamplers: full fill and partial fill

In a fixed-loop design the loop is a separate piece of tubing on the valve and the needle connects to the valve's sample port, so the loop is the only part of the sample path that sees column pressure. There are two ways to fill it.

**Full-loop (complete) filling.** You load an excess of sample so that the loop contains nothing else; the loop sets the volume. Rheodyne recommends two to five loop volumes of excess and quotes a typical reproducibility of 0.1% RSD for loops of 5 µL and larger, while warning that accuracy is limited because loop volumes are nominal ([Rheodyne catalogue, Tech Tip #5](https://analytics-shop.com/media/pdfs/RY7030-003.PAGE_25.pdf)).

**Partial-loop filling.** The syringe sets the volume and you load no more than half the loop. The reason is laminar flow: sample flows down the centre of the loop at twice the average velocity, so more than half a loop volume starts to spill out of the far end ([Agilent, partial loop filling](https://lc.help.agilent.com/en/4402148619-2-1-171136788883294731-20.htm)). The trade-offs between the two modes, with loop-sizing examples, are in [full-loop vs partial-loop injection](/en/blog/full-loop-vs-partial-loop-injection).

### Pull-to-fill and push-to-fill

- **Push-to-fill.** The syringe first draws sample into the needle or a holding line, then reverses and pushes it through the valve into the loop, as a chromatographer does by hand with a manual injector. In partial fill the loop receives what the syringe dispensed, provided the connecting passage between needle and loop is accounted for. Rheodyne showed why that passage matters: with a loop filler port, the first microlitres dispensed never reach the loop and the first sample that does arrive is diluted ([Rheodyne Technical Note 5](https://www.idex-hs.com/docs/default-source/tech-notes/tech_note_5.pdf-%283%29.zip?sfvrsn=e4ca6d90_3), Figure 5). A variant sandwiches the sample between plugs of transport solvent so that nothing is wasted; Spark Holland calls this microliter pickup and notes it is more prone to dispersion because both ends of the sample plug touch solvent ([Spark Holland white paper](https://www.sparkholland.com/wp-content/uploads/2024/02/The-comparison-of-LC-autosampler-injection-designs.pdf)).
- **Pull-to-fill.** The syringe sits beyond the loop and sucks sample from the vial through the needle, the transfer line and the valve into the loop. The plumbing is simple and the gradient delay volume is low, but the needle and line must fill with sample before any reaches the loop. Thermo Fisher sums up pulled-loop samplers as low in delay volume but prone to wasting excess sample, with lower volume precision and more carryover ([Thermo Fisher SP74151](https://documents.thermofisher.com/TFS-Assets/CMD/brochures/sp-74151-lc-ms-vanquish-neo-low-flow-injection-sp74151-en.pdf)). As an example of the overfill a fixed-loop autosampler can need, the partial-loop-with-needle-overfill mode of Waters' ACQUITY UPLC Sample Manager draws the specified volume plus 15 µL by default (14 µL before the sample, 1 µL after) ([Waters WKB11277](https://support.waters.com/KB_Inst/Chromatography/WKB11277_How_much_sample_is_consumed_using_the_partial_loop_with_needle_overfill_injection_mode_on_the_ACQUITY_UPLC_Sample_Manager)).

> [!TIP]
> Suction can never exceed about 1 bar, while a syringe can push at several bar. With viscous or volatile samples, pull-to-fill designs are the first to form bubbles: slow the draw speed first.

## Flow-through needle and split-loop designs

In these designs the needle is part of the sample loop, and needle, loop and metering device stay in the high-pressure flow path during the run. Agilent calls its version flow-through injection and Thermo Fisher calls its version split loop; details differ, but in both the needle is part of the high-pressure flow path. Agilent's Hybrid Multisampler sequence shows it ([Agilent, optimum delay volume](https://lc.help.agilent.com/en/9007203058107915-1-2-171136788883294731-20.htm)):

1. Valve in main path: mobile phase flows from the injection valve through the metering device, needle and needle seat to the column.
2. Valve to bypass: the metering device draws sample from the vial into the needle.
3. Needle wash, then the needle returns to its seat.
4. Valve back to main path: the pump flow pushes the sample onto the column.

The benefits: everything you draw is injected, and mobile phase flushes the needle and loop during the run. Thermo Fisher specifies its Vanquish Split Sampler HT and FT at below 0.25% area RSD for 1 µL injections, with 2 µL of sample needed for a 1 µL injection ([Vanquish specifications, PS73319](https://documents.thermofisher.com/TFS-Assets/CMD/Specification-Sheets/ps-73319-hplc-vanquish-autosampler-ps73319-en.pdf)).

The costs: the needle-to-seat joint must hold full system pressure every time the needle returns (the Split Sampler HT for Vanquish Horizon is specified to 151 MPa, about 22,000 psi), and the needle, loop and metering path add gradient delay volume for as long as they stay in the flow.

## How do the injection designs compare?

| | Fixed loop, full fill | Fixed loop, partial fill | Flow-through needle or split loop |
|---|---|---|---|
| What sets the volume | Loop | Syringe or metering device | Metering device |
| Sample drawn but not injected | 1-4 loop volumes of overfill, plus the line | Needle and line volume (pull-to-fill) or little (push-to-fill) | None by design |
| Precision | Highest | Set by the syringe drive | Set by the metering device |
| Parts at column pressure | Valve and loop | Valve and loop | Valve, loop, needle, needle seat, metering device |
| Extra gradient delay volume | Loop, while in INJECT | Loop, while in INJECT | Needle, loop and metering path, unless switched out |

Spark Holland tested all of these on one platform and found full loop the most reproducible, partial loopfill and split loop comparable to it, and full loop, partial loopfill and split loop comparable in dispersion ([Spark Holland white paper](https://www.sparkholland.com/wp-content/uploads/2024/02/The-comparison-of-LC-autosampler-injection-designs.pdf)). Carryover depends less on the principle than on which surfaces touch the sample and how they are washed; see [autosampler carryover](/en/blog/autosampler-carryover).

## Gradient delay volume: why the autosampler's share matters

Gradient delay volume (dwell volume) is the volume from the point where the mobile phase is mixed to the head of the column ([Thermo Fisher SP74151](https://documents.thermofisher.com/TFS-Assets/CMD/brochures/sp-74151-lc-ms-vanquish-neo-low-flow-injection-sp74151-en.pdf)). Until the gradient has travelled through it, the column sees the starting composition. Vendors publish the autosampler's share ([Agilent](https://lc.help.agilent.com/en/9007203058107915-1-2-171136788883294731-20.htm), [Thermo Fisher PS73319](https://documents.thermofisher.com/TFS-Assets/CMD/Specification-Sheets/ps-73319-hplc-vanquish-autosampler-ps73319-en.pdf)):

| Autosampler (vendor data) | Autosampler delay volume |
|---|---|
| Agilent 1290/1260 Infinity III Hybrid Multisampler G7137B, flow-through mode | about 78 µL |
| Agilent Hybrid Multisampler G7167C, flow-through mode | about 265 µL |
| Thermo Vanquish Split Sampler HT/FT, 25 µL loop (10 µL loop) | 110 µL (83 µL) |
| Thermo Vanquish Split Sampler C/CT, 100 µL loop (10 µL loop) | 255 µL (124 µL) |

A worked example: a 2.1 × 50 mm column has a void volume of roughly 118 µL (π × 1.05² × 50 × 0.68). At 0.4 mL/min (6.7 µL/s), a 265 µL autosampler delay holds the column at starting conditions for 40 s, about 2.2 column volumes, before the gradient arrives. On a 4.6 × 150 mm column (void volume about 1.7 mL) at 1.5 mL/min, the same 265 µL is 11 s and 0.16 column volumes: negligible. Delay volume matters for narrow-bore columns, low flow rates, short gradients and method transfer. That is why Agilent can switch the valve to bypass a few seconds after injection (Automatic Delay Volume Reduction) and why the Vanquish Neo can take its loop offline for nano-flow work.

In a fixed-loop design the loop counts towards the delay volume only while the valve is in INJECT. If the autosampler returns to LOAD after the flush in step 7, the loop is out of the gradient path for the rest of the run.

## Where the Elutrolab AS-48 fits

Elutrolab makes the [AS-48](/en), a compact autosampler that is still an engineering prototype. It is a fixed-loop, partial-fill design built around three choices:

- **Fixed 240 µL loop, 1-100 µL injections.** The largest injection stays below half the loop, inside the linear region of partial filling. An optional 25 µL loop covers 1-5 µL.
- **Backflush to the column.** The sample leaves the loop through the same end it entered, so a 10 µL plug does not have to travel through the remaining 230 µL of loop to reach the column.
- **Low-pressure needle.** The needle, syringe and sample line never see column pressure; only the loop is switched into the high-pressure stream. No high-pressure tube flexes while the needle moves, and there is no needle seat that has to seal at column pressure.

The valve has a 5000 psi (345 bar) design rating*, and the cycle time starts from 30 s for a 1 µL injection at 2 mL/min*. Values marked * are design targets to be confirmed by testing. Full figures are in the [AS-48 specifications](/en#specifications).

> Note from Elutrolab: The Elutrolab AS-48 is a fixed-loop autosampler: a 240 µL loop with backflush to the column, 1-100 µL partial-fill injections and a needle line that never sees column pressure. It is an engineering prototype; values marked * are design targets.

## Which autosampler design should you choose?

- **Plenty of sample, one volume, best precision:** fixed loop, full filling.
- **Variable volumes:** fixed loop, partial filling, loop at least twice the largest volume.
- **A few microlitres per vial:** flow-through needle or split loop.
- **Narrow-bore columns, fast gradients:** check the published delay volume, or take the loop out of the flow after injection.
- **Compact analyzers and reactor setups:** a low-pressure needle keeps moving parts away from column pressure; see [building an autosampler into your instrument](/en/blog/oem-autosampler-integration) and our comparison of [compact HPLC autosamplers](/en/blog/compact-hplc-autosamplers).

## Frequently asked questions

### How does an HPLC autosampler inject a sample?

It draws sample from a vial with a needle and a syringe or metering device, places it in a sample loop, then switches an injection valve so the pump flow carries the loop contents onto the column. The needle and sample path are washed before the next vial.

### What is the difference between a fixed-loop and a flow-through needle autosampler?

In a fixed-loop design only the loop is switched into the high-pressure flow and the needle stays at low pressure. In flow-through needle and split-loop designs the needle and loop stay in the flow path, so everything drawn is injected, but the needle seat must seal at full system pressure and the needle and loop add gradient delay volume.

### Why can a loop only be filled to about half its volume in partial-fill mode?

Laminar flow gives the liquid a parabolic velocity profile: the centre moves at about twice the average speed, so the sample front reaches the far end of the loop after roughly half a loop volume. Beyond that, sample is lost to waste and accuracy and precision fall.

### How much gradient delay volume does an autosampler add?

It depends on the design. Agilent quotes about 78 µL and 265 µL for two Hybrid Multisampler models in flow-through mode, and Thermo Fisher quotes 110 µL for a Vanquish split sampler with a 25 µL loop. On a 2.1 × 50 mm column that can equal one to two column volumes.


## Sources

- [IDEX Health & Science: Fluidics FAQs (what an injection valve does)](https://www.idex-hs.com/contact/contact-us/faqs/fluidics)
- [IDEX Health & Science: Injectors troubleshooting guide](https://www.idex-hs.com/resources/resources-detail/injectors-troubleshooting-guide)
- [Rheodyne (IDEX): Technical Note 5, Achieving accuracy and precision with Rheodyne manual sample injectors](https://www.idex-hs.com/docs/default-source/tech-notes/tech_note_5.pdf-%283%29.zip?sfvrsn=e4ca6d90_3)
- [Agilent: Partial loop filling (G1328C manual injector user information)](https://lc.help.agilent.com/en/4402148619-2-1-171136788883294731-20.htm)
- [Agilent: How to configure the optimum delay volume (Hybrid Multisampler)](https://lc.help.agilent.com/en/9007203058107915-1-2-171136788883294731-20.htm)
- [Thermo Fisher Scientific: Redefining low-flow sample injection with the Vanquish Neo autosampler (SP74151)](https://documents.thermofisher.com/TFS-Assets/CMD/brochures/sp-74151-lc-ms-vanquish-neo-low-flow-injection-sp74151-en.pdf)
- [Thermo Fisher Scientific: Vanquish split samplers product specifications (PS73319)](https://documents.thermofisher.com/TFS-Assets/CMD/Specification-Sheets/ps-73319-hplc-vanquish-autosampler-ps73319-en.pdf)
- [Spark Holland: The comparison of LC autosampler injection designs, fixed loop versus split loop (white paper, 2023)](https://www.sparkholland.com/wp-content/uploads/2024/02/The-comparison-of-LC-autosampler-injection-designs.pdf)
- [Waters: How much sample is consumed using partial loop with needle overfill on the ACQUITY UPLC Sample Manager (WKB11277)](https://support.waters.com/KB_Inst/Chromatography/WKB11277_How_much_sample_is_consumed_using_the_partial_loop_with_needle_overfill_injection_mode_on_the_ACQUITY_UPLC_Sample_Manager)