# HPLC vials, caps and septa: a practical selection guide

> How to choose HPLC vials, caps and septa: neck finishes, glass or polypropylene, inserts with real residual volumes, septum trade-offs and worked examples.

Source: https://elutrolab.com/en/blog/hplc-vials-caps-septa
Published: 2026-10-13
Publisher: Elutrolab (https://elutrolab.com)

For most reversed-phase HPLC work, the right default is a 2 mL, 12 × 32 mm vial of clear borosilicate glass with a 9 mm screw cap and a PTFE/silicone septum. Change one thing at a time when the sample demands it: amber glass for light-sensitive compounds, deactivated glass or polypropylene for basic analytes, proteins and metals, a conical insert or total-recovery vial when you have less than about 250 µL, and a different septum when the needle, the draw volume or the solvent calls for it.

## Key points

- The 12 × 32 mm body is close to universal. The neck finish (8-425, 9 mm screw, 10-425, 11 mm crimp or snap) decides which caps and inserts fit.
- Glass can leach sodium and adsorb basic compounds on its silanols. Polypropylene avoids both but can adsorb hydrophobic compounds and does not tolerate chlorinated solvents.
- Residual volume belongs to the vial and the autosampler together: Waters lists 10 µL for one sampler and 135 µL for another with the same vial.
- Pre-slit septa stop a vacuum forming during the draw but let more solvent escape: 2.2% against 0.2% acetone loss over three days in a Waters test.
- PTFE/silicone is the versatile default. PTFE/red rubber, PTFE-only discs and integral plastic caps are for single or short-interval injections.

## What is a standard HPLC vial?

The 2 mL autosampler vial is nominally 12 × 32 mm; Thermo Fisher lists the outer diameter of a 9 mm short-thread vial as [11.6 mm](https://www.thermofisher.cn/order/catalog/product/cn/en/11191021). Trays, racks and grippers are built around this body, so the body is rarely the problem. The neck is.

A finish code such as 9-425 has two parts. The first number is the approximate outer diameter of the thread in millimetres, and 425 is the thread profile and pitch that the cap must match, according to [MicroSolv's definition](https://www.mtc-usa.com/kb-article/aa-04369). The code says nothing about the inner diameter of the opening, which is what matters for inserts and needle clearance.

| Finish | Closure | Tool | What to know |
|---|---|---|---|
| 8-425 screw | 8 mm screw cap | None | Narrower mouth with its own, smaller inserts: [Agilent's](https://www.agilent.com/en/products/lab-supplies/chromatography-spectroscopy/vials-closures/inserts) conical inserts for 8 mm vials hold 200 µL at most, against 350 µL for wide-opening vials |
| 9 mm short thread | 9 mm screw cap | None | Wide-mouth screw finish, takes the larger inserts |
| 10-425 screw | 10 mm screw cap | None | Caps and septa are specific to this thread; they do not fit 8 or 9 mm necks |
| 11 mm crimp | Aluminium crimp cap | Crimper | Seal depends on the crimper setting; the cap is destroyed on removal |
| 11 mm snap | Polymer snap cap | None | Shimadzu rates snap caps' sealing as moderate against high for screw caps, and notes they are not easy to remove |

Screw caps are the most common type, according to [Shimadzu's vial guide](https://www.shimadzu.com/an/service-support/technical-support/liquid-chromatography/tips_and_tricks/vials-1.html). Buy caps for the finish, not for "2 mL vials" in general, and do not mix vial and cap systems without testing the combination.

## Glass, deactivated glass or polypropylene?

Clear autosampler vials are usually Type I (first hydrolytic class) borosilicate glass. MicroSolv distinguishes ["33 expansion" glass (Type I, Class A), formed at about 1,200 °C, from "51 expansion" glass (Type I, Class B), formed at about 1,000 °C](https://www.mtc-usa.com/kb-article/aa-00209). It treats both as suitable for chromatography. For the analyst, the surface matters more than the glass class.

Two surface effects cause most vial-related errors, both described in [Shimadzu's note on vial interactions](https://www.shimadzu.com/an/service-support/technical-support/liquid-chromatography/tips_and_tricks/vials-2.html):

- **Sodium leaching.** Sodium leaves the glass once solvent is added. It can raise the pH of the sample and, in LC-MS, form sodium adducts. With 5 mg/L meloxicam left for 48 h, sodium adducts accounted for more than 50% of the analyte ions in some vials, which costs sensitivity if you quantify the protonated ion.
- **Adsorption of bases on silanols.** Basic compounds bind to surface silanols by ion exchange, quickly at first and then more slowly. Shimadzu tracked chlorhexidine at 1 mg/L over 24 and 72 h and notes that the loss grows as concentration falls, so it shows up as poor recovery and a curved calibration at the low end.

Deactivated (silanised) glass is chemically treated to reduce surface activity and is sold for [basic and surface-active compounds](https://kb.mtc-usa.com/product-details/id/4125080/catalog/9502s-e-0dcv). Polypropylene has no silanols at all, which helps [proteins, peptides and basic compounds](https://www.mtc-usa.com/kb-article/aa-01553), and it releases no metal ions, which is why Shimadzu calls it essential for ion chromatography.

| Vial material | Choose it for | Watch out for |
|---|---|---|
| Clear borosilicate glass | General LC, organic solvents, storage | Sodium leaching, adsorption of bases at low concentration |
| Amber borosilicate glass | Light-sensitive analytes | Shimadzu notes amber glass does not give complete shading; keep the tray dark too |
| Deactivated (silanised) glass | Basic or adsorption-prone analytes in organic or mixed solvents | Higher cost; verify recovery rather than assume it |
| Polypropylene | Proteins, peptides, basic compounds, ion chromatography, metals | Hydrophobic adsorption; additives can add [extractables and LC-MS background](https://www.mtc-usa.com/kb-article/aa-01553); Thermo rates polypropylene "not advisable" with chloroform, dichloromethane, benzene and ether |

The polypropylene ratings come from the chemical resistance chart in [Thermo Fisher's septa guide](https://documents.thermofisher.com/TFS-Assets/CMD/Flyers/fl-73838-cc-septa-selection-guide-fl73838-en.pdf). If in doubt, leave your lowest standard in two vial types for 24 h and compare peak areas; Shimadzu advises finding adsorption and leaching during method development, not after validation.

## Inserts, micro-vials and total-recovery vials

When sample is scarce, the question is how much liquid the needle cannot reach. Makers publish this as residual volume:

| Product (maker data) | Usable or recommended volume | Residual volume |
|---|---|---|
| [Agilent](https://www.agilent.com/en/products/lab-supplies/chromatography-spectroscopy/vials-closures/inserts) conical glass insert with polymer feet, wide-opening vials (5181-1270) | 250 µL recommended, 350 µL max | < 3 µL |
| Agilent flat-bottom glass insert, wide-opening vials (5181-3377) | 400 µL recommended, 440 µL max | < 25 µL |
| Agilent conical insert, 8 mm screw-top vials (8010-0124) | 150 µL recommended, 200 µL max | < 2 µL |
| [Thermo SureSTART](https://documents.thermofisher.com/TFS-Assets/CMD/Flyers/fl-002900-ccs-surestart-total-recovery-fused-insert-polyspring-vials-fl002900-na-en.pdf) fused-insert vial | 25-250 µL | 2 µL |
| Thermo SureSTART total-recovery vial | 25 µL to 1.5 mL | 1 µL |
| [Waters Total Recovery](https://support.waters.com/KB_Chem/Other/WKB112520_What_is_the_maximum_residual_volume_of_my_Total_Recovery_vial_part_186000384C) vial (186000384C) | Not stated | 4 µL (ACQUITY I-Class, fixed loop) to 10 µL (ACQUITY H-Class, flow-through needle) |
| [Waters Max Recovery](https://support.waters.com/KB_Chem/Sample_Preparation/WKB247278_What_is_the_maximum_residual_volume_of_Max_Recovery_Vials) vial | Not stated | 10 µL (2707/2777) to 135 µL (ACQUITY flow-through needle) |

In the same Agilent range, the conical insert's residual limit (< 3 µL) is an eighth of the flat-bottom one (< 25 µL). Waters states that residual volume "depends on what type of injector/autosampler it is used with", so a figure from one sampler does not transfer to another. Inserts must also match the mouth: the wide-opening and 8 mm ranges are different parts. Polymer feet under the insert give a little when the needle reaches the bottom, which Agilent says reduces breakage and residual loss. Thermo adds that bubbles in small, water-rich samples spoil volume reproducibility, so tap the vial or spin it briefly before loading.

### Worked example: how much sample must you load?

```text
V_load = n × V_consumed + V_residual + V_margin
```

Suppose you need three injections of 10 µL, your autosampler consumes exactly the injection volume each time, and you want a 20 µL margin. For a plain flat-bottom 2 mL vial, the residual is the cylinder of liquid under the needle tip. Assume an internal diameter of 10 mm and a needle that stops 2 mm above the floor (check both for your own vial and sampler):

```text
V_residual ≈ π × (10 / 2)² × 2 ≈ 157 mm³ = 157 µL
```

| Container | V_residual | V_load for 3 × 10 µL |
|---|---|---|
| Plain 2 mL vial (assumed geometry above) | about 157 µL | about 207 µL |
| Waters Max Recovery on ACQUITY flow-through needle | 135 µL | 185 µL |
| Agilent conical insert with polymer feet | < 3 µL | about 53 µL |
| Thermo total-recovery vial | 1 µL | about 51 µL |

With 60 µL of sample, only the conical insert and the total-recovery vial give you three injections. If your autosampler draws more than it injects (flush volume, air gaps, or overfill for full-loop injection), replace V_consumed with what the method really takes; the [full-loop vs partial-loop guide](/en/blog/full-loop-vs-partial-loop-injection) and the post on [how much you can inject](/en/blog/hplc-injection-volume) show how to work that out.

## Septa: materials and trade-offs

Most LC septa are two layers: a PTFE film facing the sample and an elastomer behind it for sealing. Once the needle has pierced the film, the elastomer meets the solvent. The table summarises [Thermo Fisher's septa guide](https://documents.thermofisher.com/TFS-Assets/CMD/Flyers/fl-73838-cc-septa-selection-guide-fl73838-en.pdf):

| Septum | Reseals? | Strengths | Limits | Use it for |
|---|---|---|---|---|
| PTFE/silicone | Yes, highly | Most versatile; low extractables; easy to pierce | Once pierced, standard grades are rated "use with care" for chloroform, hexane and toluene | Default for LC, repeat injections, storage |
| PTFE/silicone/PTFE | Yes, above average | Most resistant to coring; PTFE on both faces protects the silicone | Harder to pierce than soft silicone grades | Trace work, long gaps between injections, internal standards, blunt wide needles |
| Pre-slit PTFE/silicone | Yes | Easy penetration; slit releases vacuum during the draw | Slightly lower resistance to aggressive solvents; more evaporation | Thin-gauge needles, large draw volumes, samplers that need venting |
| PTFE/red rubber | Moderately | Economical; inert before puncture | Not for repeat injections with long run times, or for keeping samples once pierced | Single injections in routine runs |
| PTFE disc (0.010 in thick) | No | Best chemical inertness | Not for volatile solvents or repeat injections | One injection of an aggressive solvent |
| Polyethylene or polypropylene integral cap | No | No fluoropolymer, so Thermo lists it for PFOA work | Single use, aqueous samples | PFAS methods, one-shot aqueous samples |
| Viton | Limited | Chemical resistance similar to PTFE; recommended for chlorinated solvents | Hard, unsuitable for fine needles; costly | Last resort when other materials fail |

### How to avoid septum coring

Coring is when the needle punches a plug out of the septum. Fragments end up in the sample or inside the needle, where they contaminate the injection or block the flow path. Pre-slit septa reduce coring because the needle does not have to cut its way through, and PTFE/silicone/PTFE is the most coring-resistant laminate in Thermo's range. Hardness matters too: Thermo's soft blue silicone (ST101) is 30 Shore A, against 57 Shore A for its standard white silicone of the same 1.0 mm thickness, and it recommends softer septa for fine-gauge needles. If coring starts suddenly with a septum that used to work, inspect the needle tip for damage; the [autosampler troubleshooting guide](/en/blog/autosampler-troubleshooting) covers the symptoms.

### Why a sealed vial can starve the syringe

A non-slit septum that grips the needle tightly turns the vial into a closed volume. Boyle's law gives the pressure left in the headspace after a draw:

```text
P_after = P_ambient × V_headspace / (V_headspace + V_drawn)
```

Drawing 100 µL from a vial with 1.0 mL of headspace leaves 1013 × 1000 / 1100 ≈ 921 mbar, about 90 mbar below ambient. Overfill the vial so only 0.3 mL of headspace remains and you get 1013 × 300 / 400 ≈ 760 mbar: a quarter of an atmosphere pulling against the syringe, which then draws less than it should and may pull dissolved gas out as bubbles. Waters attributes [low first-injection areas on its flow-through-needle samplers](https://support.waters.com/KB_Inst/Chromatography/WKB89100_ACQUITY_FTN-R_first_injection_inaccurate) to non-slit septa and says pre-slit septa [prevent vacuum formation](https://support.waters.com/KB_Chem/Other/WKB264567_What_is_the_purpose_of_Pre-Slit_Septa). Shimadzu, by contrast, uses slitless septa as standard on its LC autosamplers because its needle tip is designed for them. Follow your sampler maker's recommendation.

### Pre-slit septa and evaporation

The slit that admits air also lets vapour out. Waters measured 100% acetone at 20 °C over three days: [about 0.2% evaporation with a non-slit cap and 2.2% with a pre-slit cap](https://support.waters.com/KB_Chem/Other/WKB125442_How_does_evaporation_compare_from_a_vial_with_a_pre-slit_septa_to_one_with_a_non-pre-slit_septa), rising to 1.5% and 3.2% once the septum had been pierced. Losing 2.2% of the solvent raises every concentration by a factor of 1 / (1 − 0.022) ≈ 1.022, so a standard at the end of a three-day queue reads about 2.2% high, a bias that injection precision alone will not reveal. Waters expects much higher losses with chlorinated solvents, and it advises against pre-slit septa for [extremely volatile diluents or heated samples](https://support.waters.com/KB_Chem/Other/WKB66856_What_are_the_advantages_of_a_preslit_vial_septa_compared_to_a_nonpre-slit_vial_septa).

### Septum bleed in LC-MS

Waters reports [siloxane ions in replicate LC-MS injections](https://support.waters.com/KB_Inst/Chromatography/WKB12032_Contaminant_ions_observed_in_LCMS_runs_using_pre-slit_PTFE_or_silicone_septa_vial_caps) from pre-slit PTFE/silicone septa: the first injection is clean, then solvent on the outside of the needle reacts with the top and slit of the septum. Its suggested alternatives are a polyethylene septumless cap, a PTFE septum or a low-bleed PTFE/silicone grade. Material picked up by the needle exterior is also a route to carryover; see [autosampler carryover](/en/blog/autosampler-carryover) for wash settings.

## Setting needle depth and fill volume

The needle tip must stay below the meniscus until the last draw is complete and must not hit the bottom. Most autosamplers expose this as a needle depth or height offset in the method or instrument configuration (names vary by maker). Two rules of thumb follow from the geometry above:

- In a flat-bottom vial of about 10 mm internal diameter, every 1 mm of offset leaves about π × 5² × 1 ≈ 79 µL behind. Lower the needle only as far as your tray positioning tolerance allows.
- Raise the needle on purpose when the sample has sediment or precipitate, or when you need the upper layer of a two-phase sample.

With an insert, the needle must reach into the cone. Inserts with polymer feet tolerate light contact; bare glass inserts can crack. Do not fill vials to the neck: headspace keeps the vacuum small, and liquid touching the septum wets the cap and the outside of the needle. To check that depth and volume settings work, weigh a vial before and after a set of injections; the [autosampler qualification post](/en/blog/autosampler-qualification) gives a gravimetric procedure.

## Quick selection table

| Situation | Vial | Cap and septum |
|---|---|---|
| Routine reversed phase, water, methanol, acetonitrile | Clear glass, 9 mm screw | PTFE/silicone; pre-slit if the sampler maker recommends it |
| Light-sensitive analytes | Amber glass | PTFE/silicone, tray kept dark |
| Basic drugs at low concentration | Deactivated glass or polypropylene | PTFE/silicone |
| Proteins and peptides | Polypropylene (low-extractable grade for LC-MS) | PTFE/silicone or integral cap |
| Ion chromatography, metals | Polypropylene | Polyethylene or polypropylene cap |
| PFAS | Polypropylene | Polyethylene or polypropylene cap without fluoropolymer |
| Chlorinated or aromatic solvents | Glass | PTFE disc for one injection; Viton for repeat injections (not with fine needles) |
| Volatile diluent, long queue | Glass | Non-slit PTFE/silicone, cooled tray |
| Under 250 µL of sample | Conical insert or total-recovery vial | As above for the solvent |
| Repeat injections days apart, internal standards | Glass | PTFE/silicone/PTFE |

> Note from Elutrolab: The Elutrolab AS-48 holds 48 standard 2 mL vials (12 × 32 mm) on a removable rack with an SBS footprint; it has no tray cooling. It is an engineering prototype, and values marked * on the product page are design targets.

## Before you order a case of vials

1. Check which finishes, vial heights and cap types your sampler and racks accept. The [Elutrolab AS-48](/en#specifications) rack, for example, takes 48 vials of the 12 × 32 mm format.
2. Order caps for the finish and inserts for the mouth, from the same system.
3. Inject a blank through the cap you intend to use, to see septum extractables before they appear in samples.
4. Write the vial, cap, septum and needle depth into the method, so a change of supplier is a documented change.

## Frequently asked questions

### What size are standard HPLC autosampler vials?

The standard autosampler vial holds 2 mL and measures 12 × 32 mm on the outside (about 11.6 mm actual diameter). The body fits almost every vial-based sampler; what differs is the neck finish (8-425, 9 mm screw, 10-425, 11 mm crimp or snap), which decides the cap and insert.

### Should I use pre-slit septa in HPLC vials?

Use pre-slit septa when the needle is thin, the draw volume is large or the autosampler maker recommends them, because the slit vents the vial and stops a vacuum forming. Avoid them for very volatile diluents or long queues: in a Waters test, acetone loss over three days was 2.2% with a pre-slit cap against 0.2% with a non-slit cap.

### When should I use polypropylene vials instead of glass?

Use polypropylene for proteins, peptides and basic compounds that adsorb to glass silanols, for ion chromatography and metals, and when sodium adducts from glass disturb LC-MS. Stay with glass for chlorinated or aromatic solvents and for hydrophobic analytes, which can adsorb to polypropylene.

### How much sample is left in a vial after the last injection?

It depends on the vial and the autosampler together. Maker data range from 1-3 µL for total-recovery vials and conical inserts to under 25 µL for flat-bottom inserts, and Waters lists 10 µL to 135 µL for the same Max Recovery vial on different samplers. A plain flat-bottom 2 mL vial can leave more than 100 µL, depending on how close to the floor the needle stops.

### Can I re-inject from a vial that has already been pierced?

With PTFE/silicone or PTFE/silicone/PTFE septa, yes, because they reseal. PTFE/red rubber is not recommended for multiple injections with long run times or for keeping samples after the first puncture, and PTFE-only discs and polyethylene or polypropylene integral caps do not reseal at all.


## Sources

- [Agilent: Inserts, range of options (max fill, recommended fill and residual volume)](https://www.agilent.com/en/products/lab-supplies/chromatography-spectroscopy/vials-closures/inserts)
- [Thermo Fisher Scientific: Septa selection guide (FL73838)](https://documents.thermofisher.com/TFS-Assets/CMD/Flyers/fl-73838-cc-septa-selection-guide-fl73838-en.pdf)
- [Thermo Fisher Scientific: Optimizing sample integrity, total recovery, fused insert and Polyspring vials (FL002900)](https://documents.thermofisher.com/TFS-Assets/CMD/Flyers/fl-002900-ccs-surestart-total-recovery-fused-insert-polyspring-vials-fl002900-na-en.pdf)
- [Waters WKB247278: What is the maximum residual volume of Max Recovery vials?](https://support.waters.com/KB_Chem/Sample_Preparation/WKB247278_What_is_the_maximum_residual_volume_of_Max_Recovery_Vials)
- [Waters WKB112520: Maximum residual volume of the Total Recovery vial](https://support.waters.com/KB_Chem/Other/WKB112520_What_is_the_maximum_residual_volume_of_my_Total_Recovery_vial_part_186000384C)
- [Waters WKB125442: Evaporation from pre-slit versus non-slit septa](https://support.waters.com/KB_Chem/Other/WKB125442_How_does_evaporation_compare_from_a_vial_with_a_pre-slit_septa_to_one_with_a_non-pre-slit_septa)
- [Waters WKB12032: Contaminant ions in LC-MS runs with pre-slit PTFE/silicone septa](https://support.waters.com/KB_Inst/Chromatography/WKB12032_Contaminant_ions_observed_in_LCMS_runs_using_pre-slit_PTFE_or_silicone_septa_vial_caps)
- [Shimadzu: Tips for LC and LC/MS analysis (vial selection), components of a vial](https://www.shimadzu.com/an/service-support/technical-support/liquid-chromatography/tips_and_tricks/vials-1.html)
- [Shimadzu: Tips for LC and LC/MS analysis (vial selection), interactions to be aware of](https://www.shimadzu.com/an/service-support/technical-support/liquid-chromatography/tips_and_tricks/vials-2.html)
- [MicroSolv: Differences between 33 and 51 expansion glass](https://www.mtc-usa.com/kb-article/aa-00209)
- [MicroSolv: Polypropylene vials and protein adsorption compared to glass](https://www.mtc-usa.com/kb-article/aa-01553)