Separation of two peaks impossible

[michelle.byrne]

Hi, New to this forum,
I am currently trying to seperate out two peaks (butan-1-ol and IBMK). There boiling points are as follows: 116-118C and 117-118C. The oven temperature is 60C and The injector and detector temperture is 130 and 140C respectively. I lowered the oven temperature to 50C and this improved the reolution between the two peaks but they are stilled joined a little at the end. I don't think that they will pass the resolution spec. It is an old GC and the split vent is manual and I don't really know how to use it. In the method the split flow ratio states 1:50. The GC is set at 2 - 4ml/min. I am at my wits end. Please help

[GasMan]

Michelle,

Before any of the experts can help you, they will need to know the following.

1. What column are you using.

2. What carrier gas are you using.

3. A complete list of the compounds in your sample, even the ones that you are not trying to measure.

Gasman

[Schmitty]

What model is the GC? If you are expected to run a split, the split should always be "on," therefore you just need to set the split once and leave it.

Is it out of the question to go to 40°C? Are you running isothermally at 50 or 60?

[chromatographer1]

I assume the following:

You are using a methyl silicone column separating components according to an established method you are not able to modify other than small changes in temperature and flow.

Since it has worked previously.............

have you changed brands of column?

length? ID? phase thickness? actual amount of sample injected or the actual split ratio?

have you tried to increase your injector temperature?

have you checked the column inlet installation?

is the column an old column? tried a new one?

this ketone and alcohol can be separated without problems but if you cannot change column in the method you are stuck in addressing other issues.

kindly share more information. As you have noticed, without adequate information supplied it is difficult for the forum members to assist you.

best wishes,

Rod

[michelle.byrne]

Column: Supelco: SPB-1 Fused Silica Capillary Column 30m x 0.25mm x0.25um
GC Make: Shimadzu GC-14A

Chemical Components at the moment: 50:50:50 misture of Butan-1-ol: IBMK: n-buty acetate
Butan-1-ol RT = 3.426
IBMK RT = 4.386
n-butylacetate = 6.51

Carrier gas = Helium
Hydrogen and air for flame
Nitrogen to pass therough a packed column on the system to stop it burning out while the capillary column is in use.

stated method oven temp = 60C isothermally
I have lowered this down to 40C and the trought between the butna-1-ol and IBMK peaks is closer to the baseline.

The detector and injector temp is not mentioned in the paper so:
I read a paper which stated that the detector temp should always be 150 to prevent water vapour. Is this true??
I noticed that lowering the injector temperature to 130C (highest boiling point of three components is 119C n-butylacetate) has reduced tailing of all the peaks.

All suggestions welcome Please help

Michelle

[MikeD]

If I intepret your information correctly there are one or two things that might conspire against you.

Firstly as Chromatographer1 says there should be no problem with this separation, although I always see n-butanol tail a little bit on methylsilicone. It's always possible that there is something badly wrong with your flow path - leaks and so on - difficult to help you there but here are some things you could check out.

Are you saying your column flow is 2-4 ml/min? That is 75-130 cm/sec with your column dimensions. Why are you not at a more typical 30-40 cm/sec for helium ? What is your column inlet pressure ? There are little software routines that calculate mean carrier velocity. Don't know about Shimadzu but Agilent and PE have them. If you really are at the higher flow it will degrade performance and cause you to use lower temperatures than desirable.

Then there is the film thickness. Maybe not much you can do about it but butanol would prefer 1 um. Are you overloading the column? Any more than about 30 ng on your 0.25 um film thickness column will give more distortion than on a 1 um film. At 50:1 split this is equivalent to 1.5 ug. Are you injecting much more than this?

If you could make these changes you would run at a higher temperature which is slightly worse for resolution, although trivial since they elute so far apart and you are closer to carrier velocity optimum, but it's better for tailing.

[GOM]

Hi Michelle,

Could you just confirm a few thing for us

1. Would you just clarify what you mean by a 50:50:50 mix?
2. What is the volume flow coming out of your split vent?
3. What time does it take (in seconds) for butane to come out? Establish this by putting your syringe needle into the flame nozzle of a disposable lighter, press down the gas release bit and take about 5ul to inject. From this we can work out if your gas linear velocity is at a sensible value - say 20 - 40cm/sec. Your comment about a flow of 2-4mls/min concerned me slightly.
4. What size syringe are you using?
5. If your chromatogram is on a paper output you can take a picture with a digital camera and post it up.

Regards,

Ralph

[chromatographer1]

michelle byrne

If you have not checked your flow rate by now, you should have.

You should adequate separation.

best wishes,

Rod

[promochrom]

Your column is non polar. The separation is maily based on volatility. So it is difficult for these two compounds. You may try a polar column (such as HP-Wax, or Innowax. Now the separation is based on both voaltility and polarity. You should have better chance for the separation.

[sadsal123]

I am using an Elite-624 capillary column (30m * 250um * 1.4um film thickness) and I can separate these fine.

Salma

[chromatographer1]

Both the previous posters are correct. There are many other columns that will separate these two analytes. But if you are constrained by a method which has been validated previously, you may be required to determine why the separation is not achieved under the present conditions.

Please let us know how things work out.

best wishes,

Rod

[sadsal123]

hi, i m not at work at the moment but will check the optimum flow rates for you on monday.

Salma

[michelle.byrne]

" Are you saying your column flow is 2-4 ml/min? That is 75-130 cm/sec with your column dimensions. Why are you not at a more typical 30-40 cm/sec for helium ? What is your column inlet pressure ? There are little software routines that calculate mean carrier velocity. Don't know about Shimadzu but Agilent and PE have them. If you really are at the higher flow it will degrade performance and cause you to use lower temperatures than desirable. "

I usually operate automatic Agilent GC's where chemstation software is used to input variables. However, the shimazu is a manual injection GC using a portal integrator. The Carrier and make-up flow of the Helium is set manually using dials at the side of the instrument. The flow of the Hydrogen and Air is also set this way. It is not a calibrated instrument as there is no need. The flow is read in Kg/cm2 and the at the moment, I found that the following operating conditions are ok; Carrier Helium: 1.0kg/cm2, Make-up Helium: 0.5kg/cm2, Injector temperature: 130C, Detector temperature: 170C, Column temperature: 45C. There is still some tailing on all three peaks and the trought between the IBMK and n-butanol peak is closer to the baseline at these conditions.
I don't have much experience with split flow and the method states 1:50. I looked at the split flow vent and this is manual. There is a glass flute with a tube at the bottom. I have filled it with water and connected it up to the vent to count the number of bubbles traveling out of the vent but none appeared!!!!! It is set at 2-4ml min which is stated on a sticker on the instrument. But I haven't confirmed this yet. But I don't think that this will make much difference to the resolution and tailing of the peaks??? I have no way of measuring the flow of the carrier gas.

I cannot change the column. Basically I am trying to replicate the results of another experiment. However, in the paper some GC parameters were left out (i.e. det & inj & flow rate) and also factors in performing the experiment (i.e. type of filter to filter sample - samples are solidifying) were also left out. What looks like a basic experiment has turned into a nightmare.

I am injecting 0.1ul onto the column and the peaks are sharp and slender before tailing. I need to pass the USP resolution spec and tailing spec.

"1. Would you just clarify what you mean by a 50:50:50 mix?"

Sorry: 1ml:1ml:1ml n-butanol: IBMK: n-butyl acetate

2. What is the volume flow coming out of your split vent?
2-4ml/min I think

3. What time does it take (in seconds) for butane to come out? Establish this by putting your syringe needle into the flame nozzle of a disposable lighter, press down the gas release bit and take about 5ul to inject. From this we can work out if your gas linear velocity is at a sensible value - say 20 - 40cm/sec. Your comment about a flow of 2-4mls/min concerned me slightly.

I don't understand this -

4. What size syringe are you using?
1ul syringe


Thanks for all your comments and help to date. Really appreciated.

[Bruce Hamilton]

"The Carrier and make-up flow of the Helium is set manually using a dial at the side of the instrument. The flow of the Hydrogen and the Air gasses is also set this way. It is not a calibrated instrument as used there is no need. The flow is read in Kg/cm2 and the at the moment I found that the following conditions are ok, Carrier Helium: 1.0kg/cm2, Make-up Helium: 0.5kg/cm2, Inj: 130C, Det: 170C, Col: 45C.

I don't have much experience with split flow and the method states 1:50. I looked at the split flow vent and this is manual. There is a glass flute with a tube at the bottom. I have filled it with water and connected it up to the vent to count the number of bubbles traveling out of the vent but none appeared!!!!! So I know it is set at 2-4ml min which is what it is set at. But I don't think that this will make much difference to the resolution and tailing of the peaks. I have no way of measuring the flow of the carrier gas.

OK. Lets sort out your choice of music first. The glass flute is probably a soap film bubble meter. You don't fill it with water, but add a few mls of soap solution to the bulb at the bottom and sweeze the bulb to put a single bubble into the flow from the split vent or septum purge. You then time the bubble to go from the zero to reach the 1, 10, or 100 ml mark. Adding water probably would just block the flow.

You should be able to connect tubing to the flute to get it to measure flows from the detector exit . If you can, then the carrier flow alone from the detector should be about 2 - 3 ml/min, and the flow from the carrier split vent 50 - 150 ml/min.

With luck, setting the detector gases ( air hydrogen, maybe nitrogen makeup pressure to the conditions in the manual will be satisfactory.
But you can check them once you have a soap film meter on the detector outlet. Don't let any soap film or soap solution get into the detector by sweezing the bulb to quickly.

I'm assuming Shimadzu split system is similar to Agilent, in which case if your split vent flow is too low for the head pressure, too much sample will reach the column and your peaks will merge. Overloading would explain your problem.

If noboby from the forum offers detailed instructions for your instrument, and the manual isn't clear, you may want to contact the local Shimadzu technical support on the phone just to clarify which settingsare normal.

Sort out the split ratio, and the music will be much better.

Bruce Hamilton

[GOM]

Hi,

Sorry I didn't make it clear. What I was trying to do was to get you to inject an inert gas like butane onto your column. This will not be held back significantly by the phase so it will travel along at same speed as the carrier gas. So, from the time it takes from injection to appearing as a peak and knowing the length of your column, we can calculate the linear velocity of the carrier gas i.e. column length in cm/time in seconds. This should be about 20 to 40cm/sec for best efficiency. A convenient source of butane is a disposable cigarette lighter and the nozzle at the top has been designed to fit a GC syringe needle You can use this method if you find it difficult to measure the flow directly at the detector. If you are measuring the flow at the detector then remember to shut off the air, hydrogen and make up flows.

From what you say it would appear that you have a column flow of 1.3ml/min or 30 cm/sec if your pressure is 1kg/scm (14.2psi) and your oven is 40°C, which is fine. As others have pointed out it would also appear that you haven't got a 50:1 split. You should get about 65mls/min from your split vent.

If you go to these sites you can download useful flow calculation software. Try playing with the values to see what effect it has on the linear velocity.

http://www.chem.agilent.com/cag/servsup ... s/GCFC.htm#
or here
http://www.unichrom.com/dle.php

And don't worry about saying that you don't understand.

I also hope that you are injecting less than 0.1ul.

Regards,

Ralph