Step by Step Septa Tutorial Use Guide – Here’s How and Why

There are multiple variations of septa you can choose from, and it can be overwhelming. So how can you choose the right one with so many septa on the market? In this article, you will learn about the types of septa that exist on the market. Also, find out how to use each septa in an appropriate environment.

General Chromatography Vials and Applications in the Headspace

First, let’s find out what septa is in short terms: Septa is a circular silicone or silica gel gasket.

You can put septa in the cap of the HPLC Vial to seal the vial. Also, with that action, you guarantee that there is no gap between the cap and the vial in the HPLC analysis.

Here is a list without further explanations of the septa types used today:

General Chromatography Vials: 

• PTFE/silicone,
• PTFE Natural Ruber,
• PTFE synthetic Ruber,
• PTFE/SILICONE/PTFE
• PRE-Slit PTFE/SILICONE,
• Polyethylene (PE) septa
• Polypropylene (PP) septa
• Viton

Applications in headspace:

• Butyl stopper
• PTFE/rubber septa
• PTFE/silicone septa
• PTFE/molded butyl septa
• Rubber septa
• PTFE Aluminum Foil

Let’s talk about them.

PTFE/silicone

It is high quality and a pure silicone bonded to 0.005″ thick PTFE.

Bonding is to provide a pure and inert septum that reseals after punctures.

Resealability and excellent purity are essential in most HPLC and GC applications. That is why PTFE/Silicone septa are the recommended product.

PTFE/Silicone is the most versatile septum material. It is available in various formulations to meet the needs of different applications.

You can find PTFE/Silicone septa in a regular formation. Apart from that, you can find it in a soft durometer formulation. Scientists use it for applications where needle penetration is critical.

PTFE/silicone septa are appropriate for all sorts of chromatographic applications. That is because of their:

1. very constant performance,
2. low background/blank value,
3. strong chemical compatibility,
4. efficient sealing/resealing,
5. and low penetration force.

Temperature stability chart for PTFE/silicone septa:

PTFE Natural Rubber

PTFE/natural red rubber seals for GC and HPLC are priced. Also, they have acceptable chemical characteristics.

Due to their great resealability, they are suitable for many injections. Yet, compared to PRFE/RR, the penetration is not the same. Crimp seals made of aluminum are available for natural rubber septa.

When temperatures exceed 125°C, PTFE Natural Rubber septa are the best option.

PTFE Synthetic Rubber

A very cost-effective option is the PTFE/Synthetic septa, which comes with red rubber. They are often used in standard HPLC and GC situations.

In general, you can use these septa in gas chromatography with:

1. FID (a flame ionization detector),

2. TCD (a thermal conductivity detector),

3. FPD (a flame photometric detector) detectors for routine analysis.

Before puncture, PTFE/Red Rubber has a modest resealability and high chemical inertness. In addition, regular red rubber’s low durometer provides simple needle penetration even with tiny bore GC needles.

Note: The C4000-30 and C4000-51 series are high-quality red rubber with a thin PTFE coating (0.003″).

Use high-quality medium durometer red rubber with a small (0.015″) coating of PTFE in the following:

1. C4011-1A,

2. C40911-98 series,

3. C4011-51 series,

4. C4008-1A,

5. and the C4008-98 series.

Temperature stability chart for PTFE Synthetic Red Rubber septa:

PTFE/Silicone/PTFE – T/S/T

PTFE/Silicone/PTFE is the most coring-resistant septum with preserved resealing qualities. You get that when laminating a layer of 0.003″ PTFE to either side of high purity, medium durometer silicone. 

Use the T/S/T for the most demanding applications. For example, you can use it in applications such as: 

1. ultra-trace analysis,

2. longer injection intervals, 

3. or standard internal procedures.

From the T/S/T septa benefit autosamplers with a large diameter and blunt tip needle, such as: 

1. The Agilent 1050

2. The Agilent 1090

3. The Agilent 1100

When working with solvents that destroy silicone, T/S/T can help protect both sides of the elastomer.

Temperature stability chart for PTFE/Silicone/PTFE – T/S/T septa:

Pre-Slit PTFE/Silicone

Non-slit and pre-slit septa usually come with the most similar formulations. And that said, they also share most characteristics regarding chemical and physical properties.

The best way to describe Pre-Slit PTFE/Silicone is this:

A 0.005″ PTFE layer attached to pure silicone is present on the septum.

That silicone has a cut in the middle for quicker needle penetration. Also, use it to ease the vacuum that builds up when you withdraw a large volume of sample from a vial.

This septum has chromatographic properties comparable to those of a non-slit septum. The exception is that it is somewhat less resistant to harsh solvents.

Use pre-slit septa for autosamplers with narrow gauge needles or more significant volume injections. Use them where a vacuum in the vial might be a concern.

Temperature stability chart for Pre-Slit PTFE/Silicone septa:

Polyethylene (PE) septa

Polyethylene septa are available as one-piece caps with the septum molded into the cap. The needle penetration surface is 0.01″ wide, enabling the use of tiny gauge needles.

Polyethylene septa are not resealable and are you are only meant to use them once. Also, because it is free of fluoropolymer coating, it is appropriate for PFOA testing.

There are two types of polyethylene. There is high-density and low-density polyethylene.

Here is a temperature stability chart for both of them:

High-density:

Low-density:

Polypropylene (PP) septa 

PP septa, which are chemically resistant, can be found with the following characteristics:

1. Molded into closures with 0.01″ disks,

2. Molded like single-piece caps. 

Needle penetration offers a surface that has a thickness of 0.01″. This is a perfect thickness to use in various autosamplers for HPLC.

It is not optimal to reseal these septa. And it would help if you only used them with aqueous-based sample mixes for a single injection. 

Polypropylene and polyethylene septa are different. Polypropylene septa have superior solvent compatibility, although the piercing power is somewhat higher. 

The one thing that is the same for polypropylene and polyethylene septa is PFOA testing. Both of them are free of fluoropolymer coating, so it is appropriate. 

Temperature stability chart for Polypropylene (PP) septa:

Viton septa

When using chlorinated solvents, use Viton septa when you need a resealable septum. For example, you will often need it for a sample matrix that assaults all other components.

Don’t use Viton septa for applications that need many injections over a long time. They are not suited for use with 32 gauge needles or high injection rates due to their inherent hardness. 

After a needle puncture, Viton has a limited capacity to reseal. Otherwise, it has similar chemical resistance as PTFE. Therefore, Viton provides the best chemical resistance for a wide range of solvents. Apart from that, Viton has an excellent piercing resistance. But, Viton is a septum of the last choice because of its exorbitant cost. 

Temperature stability chart for Viton septa:

PTFE disk septa

A solid disk 0.010″ thick, our PTFE stands better than the most aggressive solvents. At least when it comes to chemical inertness. 

Most standard gauge metal HPLC needles can penetrate the thin membrane. 

Do not use PTFE septa with the following because you can’t release PTFE septa: 

1. volatile solvents, 

2. fast cycle periods, 

3. or many injection techniques

 For GC applications, PTFE septa are rarely employed. 

Temperature stability chart for PTFE disk septa:

Applications in the Headspace

These septa frequently need high temperatures. Also, you should only use it in conjunction with appropriate headspace caps. Suitable headspace caps are crimp or screw caps.

Headspace analysis can identify volatile compounds in a liquid or solid sample. Apart from that, it also reduces column contamination. 

ND24
Vial, cap and septa

ND9
Vial, cap and septa

Deposit a small amount of the material in a septum-sealed vial, then equilibrate at a suitable high temperature.

Here is the list of headspace septa materials:

• Butyl stopper
• PTFE/rubber septa
• PTFE/silicone septa
• PTFE/molded butyl septa
• Rubber septa
• PTFE Aluminum Foil

Gray Butyl Stopper

Low-temperature (125°C) or low-pressure applications need a cost-effective septum. 

Butyl stoppers are fabulous for sealing fixed gases and low molecular weight chemicals. 

Some things aren’t compatible because they don’t provide a PTFE film barrier. These are:

1. Alkanes, 

2. Benzene, 

3. Chlorinated solvents, 

4. Cyclohexane

Temperature stability chart for Gray butyl stopper septa:

PTFE/rubber septa

PTFE/Red Rubber Septa have:

1. strong solvent resistance, 

2. resealing properties,

3. and are coring resistant. 

When you need a PTFE barrier, they are the most cost-effective option. Until the initial puncture, the PTFE face increases solvent compatibility.

Temperature stability chart for PTFE/rubber septa:

PTFE/silicone septa

PTFE/Silicone is an ideal choice for analyzing volatile organic chemicals. These analyses happen at low concentrations or during operation at higher conditioning temperatures.

Store PTFE/Silicone septa in a glass Purepak jar to ensure: 

1. minimal background, 

2. low permeability, 

3. and the best performance of any headspace septum. 

It is best to pre-fit the septa into aluminum seals to save handling before injection. 

Speta made of PTFE/Silicone has: 

1. good resealing properties, 

2. and broad chemical compatibility. 

Temperature stability chart for PTFE/silicone septa:

PTFE/molded butyl septa

This is a molded septum with a PTFE-faced core that does not extend to the septum’s margins. The PTFE central region resists a wide range of solvents well. 

The core puncture region is resistant to coring and will reseal even after many punctures. 

The black butyl outer sealing edge fits snugly on the rim of the vial, resulting in a more secure closure. 

Temperature stability chart for PTFE/molded butyl septa:

Black rubber septa

When comparing red and black rubber septa, one thing comes to mind. Black rubber septa are molded from a higher density rubber compound compared to the red one.

This septum has characteristics like the gray butyl stopper. But, the difference is with a smaller temperature range of –20 to 100°C. 

The Black Rubber septum is an affordable choice. Well, at least for applications where you desire reduced levels of vapor penetration. You should use black rubber septa with sturdier injection needles. 

Temperature stability chart for Black rubber septa:

PTFE Aluminum Foil

First of all, PTFE Aluminum foil septa is a high-temperature septum.

This septum’s aluminum backing offers:

1. an efficient vapor barrier

2. and high-temperature compatibility.

Temperature stability chart for PTFE Aluminum Foil:

What to Consider When Choosing Septa?

When deciding which septa is best, keep the following factors in mind:

• Instrument – You can use various injector needles in different instruments. In some cases, having pre-slit septa or using a material with a lower shore value can help reduce the possibility of needle difficulties. 

• Chemicals of interest – Select septa that produce a tight seal to avoid compound loss. That is critical if your compounds are volatile. 

• Solvents – some solvents used in GC and LC investigations can affect septa integrity.

A Quick Guideline for Choosing the Right Septa

You must consider many criteria to choose the right type of septa for your research. 

For example, you must have the right kind of solvent or sample. Apart from that, you have to choose between slit or non-slit design and the capacity to reseal. 

Determination of the material makeup of the septa is by these parameters. 

Also, you have to select a suitable tip diameter. Next, think about how many injections your septa must survive. Finally, you have to avoid debris, depending on your material. 

Here is a quick guideline: 

1. Natural rubber/TEF – If you have many injections and need good resealability properties. 

2. Storage temperature: 

• -40ºC/-40ºF: use natural rubber/TEF; butyl/PTFE 
• -40ºC/-40ºF: use red rubber/PTFE 
• -60ºC/-76ºF: use silicone/PTFE 

3. Silicone/PTFE – If you have a thin needle, and you need to use a thin septa 

• Silicone/PTFE, slit – If you have a thick needle, and you need to use Slit/pre-cut liner as penetration aid (HPLC) 
• Silicone/PTFE septa – If you have a critical analysis that needs a clean liner 
• PTFE/silicone/PTFE – When dealing with low coring and when you need both sided PTFE laminated liners

General Chromatography Vials: Septa Properties

Find out about four types of septa research used in general chromatography. 

Three types of rubber

Scientists use rubber in gas chromatography for routine analysis. It has a medium resealability and is chemically inert. 

Many injections or retaining samples for extra examination are not suggested. PTFE is a chemically resistant coating that, if destroyed, exposes rubber to attack

1. PTFE/red rubber 

Rubber with a low durometer enables easy needle penetration. This is a popular and cost-effective septum for general GC.

2. PTFE/rubber 

Rubber with higher hardness for use with piercing needles. In Agilent systems, the most popular and cost-effective septa for everyday GC operations.

3. Pre-slit PTFE/red rubber 

Red rubber that has been pre-slit and has a tiny (0.003″) PTFE coating. As mentioned before, this is ideal for undoing vial vacuuming or syringe needles with narrow gauges.

Five types of silicone rubber

1. PTFE/silicone – ST1, ST15, ST18, ST2 

The myriad of varieties of thickness makes PTFE/silicone ideal. In addition, it offers a PTFE protective red coating and standard medium hardness, white silicone.

2. PTFE/silicone – ST101, ST14 

• A soft silicone bonded to PTFE. The septum is excellent for instruments with fine-gauge needles because it resists coring.

• Due to its excellent purity, it’s also suitable for LC-MS and GC-MS.

3. PTFE /silicone – ST143, ST144 

A laminated silicone to PTFE. Use a flexible needle.

4. PTFE /silicone/PTFE – TST1, TST11 

• Both sides of this medium hardness septa provides a PTFE layer. With very high, mostly above average, resealing qualities, it is the one that offers the most coring-resistant properties.

• It is best to use extended injection times or internal standards. Then, use it for the most demanding applications, such as trace analysis. 

5. Pre-slit PTFE/silicone – ST1X, ST101X, ST14X 

This pre-slit septa comes with a PTFE coating and is made of superior white silicone. It is useful when a vacuum appears in the vial. It is also instrumental in situations when a syringe needle with a thin gauge is in use.

Two types of PTFE and fluoropolymers

These options offer excellent resistance to chemicals and are often used by scientists to add a layer of protection. 

 1. PTFE 

Single injections and brief sampling cycles are possible. Unfortunately, you can’t reseal this form of the septum.

2. Viton 

With limited resealability, Viton offers the highest resistance to chemicals. Therefore, for chlorinated solvents, this is a good choice.

These septa are not suited for finer-gauge syringe needles due to inherent hardness.

Integral plastic seal

They are molded as a part of the septa’s cap. 

1. Polyethylene – PE, Polypropylene – PP 

Chemically resistant, but only for one use and not resealable. 

Because it is free of fluoropolymer coating, it is appropriate for PFOA testing.

Headspace and Sample Preparation Applications: Septa Properties 

When choosing the right septa, you have to look at their properties.

Four types of butyl rubber/chlorobutyl rubber 

1. Grey bromobutyl stopper 

There is no PTFE barrier. Because of its low permeability, it’s ideal for gas sampling.

2. Black chlorobutyl 

There is no PTFE barrier. Because of its low permeability, it’s ideal for gas sampling. 

3. Grey bromobutyl/black PTFE 

It has a PTFE barrier to work with a wide range of organic solvents with minimal gas penetration.

4. Grey PTFE/black bromobutyl molded 

PTFE insert in a specially formed seal. Butyl and PTFE-faced septa have a more positive sealing surface than non-PTFE-faced septa. 

Superior sealing properties, excellent solvent resistance, decreased coring, and strong puncture tolerance. It has also improved chemical resistance by using PTFE.

Septa Hardness – What is it and for What it is Used

A durometer test is a systematic method for determining the hardness of plastics. It calculates the empirical hardness of polymers by measuring their resistance to indentation. 

The best approach for rubbers/elastomers is shore hardness. Shore hardness is widely used for softer plastics like fluoropolymers. Shore A is the expression used for the majority of septa hardness.

The results are a valuable indicator of various polymer grades’ relative resistance to piercing. Those results inform what needle you must use to breach the seal. It also tells you whether you should use a smaller gauge needle. 

Chemical Resistance 

Chemical resistance is also something you have to consider. Find out what are the effects of chemicals on plastics, as well as their impact on the glass. 

Effects of chemicals on plastics

Chemicals can have an impact on plastics:

1. strength, 
2. flexibility, 
3. surface appearance, 
4. color, dimensions, 
5. and weight. 

An assault on the polymer chain causes:

1. oxidation, 
2. functional group reactivity, 
3. and depolymerization, which results in these modifications. 

A “stress-cracking agent” is causing stress cracking. A stress-cracking agent is dissolution in a solvent. Its absorption and permeation produce softening and swelling. 

Stress cracking is a failure of plastic in the presence of certain chemicals. A chemical attack does not cause it. Instead, the company of three variables at the same time generates stress cracking: 

1. tensile stress in the plastic ,
2. its intrinsic stress-cracking sensitivity,
3. a cracking agent.

Agents can be: 

1. Detergents, 
2. surface-active chemicals, 
3. lubricants, 
4. oils, 
5. ultrapure water, 
6. plating additives such as brighteners, 
7. and wetting agents.

When mixed and/or diluted in plastic labware, certain chemicals can be harmful. Likewise, combining chemicals from two or more classes can have a beneficial or destructive impact. 

Always do a pre-test for your specific application and follow lab safety protocols. 

There is also a problem with temperature. As temperature rises, resistance to attack diminishes, resulting in product failure. 

Chemicals and their effects on glass

In most cases, borosilicate glass (clear and amber) has a high level of chemical resistance. 

Certain chemicals may etch the glass. Surface etching typically has little effect on glass’s dimensional features. But, it might release chemical components into the sample solution. 

Physical characteristics of plastic resin and septa include:

1. Appearance – Opaque, translucent, transparent, white, red/ivory, opaque grey, white/red, red/white/red, and black.
2. Temperature – MIN. and MAX. in Celsius and Fahrenheit
3. Is it autoclavable – yes/no
4. Dry heat – yes/no
5. Gamma – yes/no
6. Is it microwaveable – yes/no
7. Ethylene oxide – yes/no
8. Analytical purity – High, Medium, Method dependant
9. Fragmentation – High, Medium, Low
10. Hardness
11. Reseability

Conclusion 

Now you have in-depth explanations on choosing septa and how to do it right. 

Let’s answer some quick frequently asked questions: 

1. What is the difference between GC and headspace GC? 

The behavior of the volatile analytes differs significantly between headspace and direct injection. Most of the sample material reaches the intake system when injecting a sample into a GC inlet.

2. Which vial should a person use for HPLC? 

PTFE/Silicone septa (T/S) are the most suitable for HPLC applications. That is because of their extensive chemical compatibility and low extractables.

3. How do I choose a suitable vial? 

Here are 8 quick tips on choosing the correct vial. Of course, we already know how to select it, and so do you, but a quick reminder is never a bad idea. 

1. Make sure your autosampler is compatible.
2. Examine the vials for quality.
3. Choose the appropriate closing.
4. Consider the size of your sample.
5. Choose the fitting cap and septum.
6. For lesser sample sizes, consider using an insert.
7. Decide on the material for the vial.
8. Decide on the best technique of storage.

So, as you may have gathered, selecting the correct vial for HPLC is not always a straightforward task however, I hope these recommendations serve as a good starting point.

If you have any questions, don’t hesitate to send us a quote, our team will reply to you in 24 hours.