Chromatography Forum

Hi,

The place where I have been doing some hplc used to use a 687 uL stainless steel injection loop. These were expensive and kept breaking so they have now fitted some kind of hplc plastic tubing. I was interested if there are any downsides to using this tubing instead of the manufacturers stainless steel injection loop. the volume of the tubing is about 700 uL as well. I guess this just means the peaks will start to elute slightly later than with the 687 uL injection loop.

It is an agilent machine with an autosampler. I think it is an 1100.

Cheers

A difference in loop size of 5% has no influence on the appearance of the chromatogram.

The plastic is probably Peek (poly-ether-ether-ketone). Its pressure resistance is limited (200 bar, maybe 350 bar). It should not be used with metallic fittings but plastic "fingerthight" fittings are needed. The original Peek is not resistant to dichloromethane, THF, dimethyl sulphoxide, conc. nitric acid, and conc. sulphuric acid (of course depending on the content of these compounds in the mobile phase and on the pressure). There are other Peek qualities available with maybe better properties.

Veronika R. Meyer

Even if the volume difference between the loops was higher you wouldn't notice any retention time difference as your compounds would be re-focused at the head of your column. Only unretained compounds would have been affected.

As mentioned above there are not downsides other than mentioned above. On the contrary there are some advantages if you are analyzing compounds that can interact with chelating metals.

I am supriced to hear that your stainless steel injection loops were breaking like this. I never experience stainless steel injection loop breaking (but I never used such large loops neither).

There is no reason that you have to use PEEK fittings on PEEK tubing. Standard steel compression screws and ferrules work quite well.

Laudrup, do you mean that you had trouble with the steel ferrules? One would have to sharply bend the tubes back and forth many times to break them. To avoid ferrule problems (cutting of tube after leaks, etc.) we use Upchurch (something different now?) Litetouch, these you can also readjust (positionally) to adapt to different Rheodyne or other systems.

There is one downside to steel fittings on polymeric tubing: too heavy a hand will collapse the i.d. of small tubing. Just be careful and they will work great together.

A common explanation to "increased back-pressure in the system" is actually to tightly screwed PEEK fingertights that are fitted on PEEK tubing.

That does not mean that we recommend steel ferrules on a PEEK tube. On the contrary, instead it is a good habit to cut a small piece from the PEEK tubing end each time it is disconnected or moved.

Hi,

Thanks you for your replies.

I don't have a lot of experience with HPLC but if the injection loop volume was greater wouldn't the retention times differ?

I am using a brownlee aquapore column and guard column with C4 packing. I am using buffers:

A: 10% MeCN / 90% Milli Q water / 0.1% TFA
B: 90% MeCN / 10% Milli Q water / 0.1% TFA

flow rate: 0.8 ml/min

injection volume: 100 uL.

My gradient is:
Time (minutes) %B
0 0
4 0
6 28
11 48
14 55
15 58
18 60
18.01 0

If the injection loop volume is greater wouldn't it take longer for the more concentrated MeCN to get to the column and hence longer for the proteins to elute?

I have just ran some more samples and the compounds are eluting about 0.5-0.6 minutes later, with the peek tubing injection loop, than they used to with the stainless steel injection loop. For example one major compound was eluting at ~8.9 minutes (with stainless steel injection loop) and now it is eluting at ~9.5 minutes. We have made up several lots of buffers and we keep getting the same results.

This seems too big a difference. Even if it was due to the different size injection loop, the retention time should only differ by: 13 uL / 800 uL/min = 0.016 min.

Does anyone have any ideas?

Cheers,
Laudrup

If you would hook up the inj. valve such that the mobile phase flow is backward to that of the injection flow direction you would not, theoretically, get a time delay due to loop volume (in partial filling). Practically, you may get some problems due to laminar flow (discussed elsewhere).

On the real cause for the delay: Did you check (measure) the flow of the runs with the different loops? Did you change the mobile phase, or other parameters, concurrently?

The only thing we changed was the injection loop. We didn't measure the flow of the runs with the different loop. I don't think it has anything to do with the mobile phases since we have made up several different lots of buffers from different lots of Acetonitrile.

laudrup,

looks like an interaction between your analyte and the peek material.

laudrup, I was thinking of a small flow change caused by differences in flow resistance due to differences in the loop joints (connections).

Laudrup,

Has the system pressure changed since you changed from using the steel loop to the PEEK loop?

laudrup,

Indeed, you are correct that injection volume differences will result in apparent retention time differences assuming you are making injections under focusing condition. Strictly speaking, this difference should be corrected for in order to report the actual retention time but in general it doesn't seem to be worth the extra effort necessary to determine the proper correction factor. But this brings up another possibility no one has mentioned yet: perhaps the volume of one of these loops is incorrect. For PEEK tubing the variability of ID is fairly substantial so the nominal injection volume you mention (which is most likely based on a length measurement with an assumption as to the internal diameter) might differ considerably from the actual injection volume. If you have access to a good analytical balance (i.e. one that reads to a 10th of a milligram), you can quickly determine the volume of your loop by weighing it empty and weighing it filled with water.