Chromatography Forum

Hi, I looked at the flows that you are using and for Helium, 2-4ml/min is too high. If your start temp is 40oC, your optimum temperature will be 0.7-1.9 ml/min. Try about 1ml/min and see if your resolution/separation is better. Let us know how you get on.


Good luck,

Salma

Hi all,
Thanks for the help so far.

I will post up progress and summary so far of the method:


Stated Method details

Column Details: SPB-1 Fused Silica Capillary Column (30m x 0.25mm x 0.25um) New column and cannot cahnge it

Oven Temp = 60C Isothermally
Spilt Ratio = 1:50


Progress so far

GC Make & Model: Schimadzu GC-14A - Manual injection
Column Details = as above
Column Make = Supelco
Syringe Size = Glass 1ul
Sample size injected = 0.1ul

chemical components =
Butan-1-ol, 116 -118C boiling point (bp), Retention time (RT) = 3.78min, Area = 348877
IBMK (internal Standards), 117-118C bp, RT = 4.94mins, Area = 349099
N-butyl acetate, 126-127C, RT = 7.66mins, Area = 317879

Best GC conditions so far
Oven Temperature (Temp) = 45C Isothermally
Injection Temp = 135C
Detector Temp = 170C

Carrier Gas = Helium
GC Flame (FID, flame ionisation detector) = Air & hydrogen
Packed column on detector & injector 2. So passing nitrogen down this column

Helium Gas Flow = 22cm/sec
Split flow = 2.63mls/ min

Aim: Studying the ratio of the areas of the butanol and n-butyl acetate peaks during a reaction.

Validating the method at the moment to obtain the best possible resolution and tailing for the three peaks. At the moment n-butanol and IBMK are not seperating out correctly. There is a trought between the two peaks however it is quiet close to the baseline. There is tailing on all the peaks espically the n-butyl acetate peak.


Butane experiment
thanks a mill Ralph. This trick is really good and fast.
Injected 5ul and 10ul of butane gas onto the column and the following results were achieved:

5ul Butane: RT = 2.306mins, Area = 168947
10ul Butane: RT = 2.246mins; Area = 340904

Using the above
Velocity of carrier gas = 22cm/ sec or 2.246min/30meters, 1sec/ 0.22m, 0.22m/sec Think this is right


Gas Flow (Soap Film bubble )

Carried out this test. Thanks Bruce for explaining this.
Flow from split flow is 3.8sec/ 10mls
Which is 2.63mls/ min
The glass tube was graduated to 10mls.

I also noticed that there was two vents side by side. One vents states purge and the other vent states split. So I measured the flow from the split vent. There was no flow exiting the purge vent.

So should I alter any of the above parameters or should I just go with the peak shapes that I have.

Thanks a mill for the all the help that I have got so far on the forum. I really apreciate the help.

Michelle

Michelle

If your splitter flow is only 2.63ml/min your split ratio is not 50:1.

Increase your split flow to 50cc/min and try your injection again.

best wishes,

Rod

Hi Michelle,

Your column flow looks fine.

"Carried out this test. Thanks Bruce for explaining this.
Flow from split flow is 3.8sec/ 10mls
Which is 2.63mls/ min
The glass tube was graduated to 10mls. " how do you get this quote thing to work?!!!

Just recheck your calculation. If it takes only 3.8secs for 10mls then in 1 minute you will get 158mls. This implies that you actually have a suitably high split ratio.

The purge vent is probably the septum purge. It passes a flow of carrier across the bottom of your hot septum and carries away any septum volatiles e.g. plasticisers, rather than letting them accumulate on your column. This can be one of the causes of the ghost peaks that can appear if the instrument has not been run for a few hours. The plasticisers have condensed at the front of your cool column, just waiting to be moved by the oven temperature ramp. It is usually set to 1-2mls/min.

Just one more thing - try injecting an "empty syringe" of your sample. Take up your sample as usual but before injecting it press the plunger all the way down - bring it back up to 0.1ul then inject. It is a way of reducing the amount that you inject. You may find that it improves your chromatography. You only have a thin film and it can be easily overloaded by the almost neat solution that you are injecting. You could also increase the split ratio more. The sample capacity of your film is only 50 to 100ug.

Regards,

Ralph

Yes, 3.8 sec per 10 mL is 2.63mL/second, not minute. I knew the measurement answer was wrong, just did not do the math.

And Ralph is right in suggesting a smaller injection amount.

The other possibility is an incorrection installation of the column in the injector. It may have slipped up or down while being installed. Double check that the length of column past the ferrule is correct.

best wishes,

Rod

'''

Syringe Size = Glass 1ul
Sample size injected = 0.1ul

chemical components =
Butan-1-ol, 116 -118C boiling point (bp), Retention time (RT) = 3.78min, Area = 348877
IBMK (internal Standards), 117-118C bp, RT = 4.94mins, Area = 349099
N-butyl acetate, 126-127C, RT = 7.66mins, Area = 317879

Best GC conditions so far
Oven Temperature (Temp) = 45C Isothermally
Injection Temp = 135C
Detector Temp = 170C

Hi Michelle,

Thanks very much for the detailed post, it really helps us all offer advice. Rod and Ralph have offered good advice. The Septum Purge should be 2 - 3 ml/min, as stated above. The area counts and retention times of the peaks suggest that maybe we have two - three areas to concentrate on.

1. The amount injected, as suggested by Ralph and Rod above, you need to get it down to 0.1 ul.

If the empty syringe technique suggested by Ralph doesn't reduce the peak areas, you can try the four segment technique on your syringe.
The advantage of this technique is that a fairly slow insertion of the syringe doesn't cause the sample to boil out of the needle, but you still need to push the plunger down really quickly and immediately withdraw the whole syringe as soon as the plunger hits the bottom.

First push the syringe needle completely in then withdraw it until the air in needle is visible in the glass barrel. Then immerse it in the sample and withdraw the plunger a further 0.1ul and lift the tip out of the sample and withdraw the plunder more until the 0.1ul is visible in the glass barrel.

Then insert the syringe needle into the septum as quickly as you can, and as soon as the bottom of the barrel reaches the top of septa, quickly push the needle completely in and quickly withdraw the whole syringe.

Hopefully a quick and small injection will help reduce peak tailing, and it's only practice that achieves good injection technique.

2. The peaks may be tailing and merging because the detector gases don't have enough gas flow to keep the peaks sharp. The difference in the retention times ( over a minute ) suggests tailing is really bad. If the detector has a provision for adding "makeup" gas for capillary columns, it should be switched on and set to around 20 - 30 mls/min. The unretained n-butane should have been really sharp with no tailing. If there was tailing, I would first look at all detector gas pressures and flowrates, then the injector.

3. If the BuOAc is still tailing badly after checking the detector gas flows, I would suggest conditioning the column and injector overnight at higher temperatures ( 200C ) with all gases flowing, then increasing the injector temperature in the method to about 200C and seeing if the peaks sharpen up.

Good luck, and keep having fun,

Bruce Hamilton

Hi,

Update:

Column Installation
1. changed the septum
2. The glass liner was upside down - so changed this
3. Cut the ends of the column
4. The column lenghts I used were not correct. I checked the manual for the GC and the manual contradicts itself on these lenghts. It states the following at the start & end of manual:
Injector port column (col) lenght = 40mm or 25mm
Detector port column lenght = 75mm or 85mm
I used the 40mm injector col lenght & the 75mm detector Col lenght


Purge & Split Vent flows

Purge Vent Flow: I opened this valve and set the flow to around 2ml/min or 5sec/ 10ml

Split Vent flow: Measured this again. It is giving conflicting results. So I definetly set it this time to around 150ml/min or to be exact 3.6sec/10ml
If the method states that the split flow should be 1:50, does this mean 1ml in 50min or is it 50ml in 1min. I am getting confused with the units.
Should I adjust the split flow to 1:50 and will that allow me to operate at the higher temperature of 60C as stated in the method or should I just keep it at the 150ml/min

How to use the soap bubble meter

" how do you get this quote thing to work?!!!

A tiny bit of washing-up liquid in a beaker of about 10ml water. Pour about 1ml into rubber squichy bulb thing at the bottom and attach to the end of the glass tube. There should be a long thin plastic tube attached to the glass tube just above the rubber bulb. Attach the plastic tube to the vent and press the rubber bulb a couple of times until a bubble travels above the entrance of the plastic tube to the glass tube. The gas traveling along the plastic tube from the vent will push the bubble up the tube and when it passes the 0 mark press go on the timer until it reaches the graduated 10ml mark and then stop. thanks Bruce

Syringe
empty Syringe technique/ four segment syringe technique.
Ok I tried this but I don't really understand the instructions above for either technique.

Empty syringe technique
Do you mean take up the 0.1ul of sample and then when the syringe is out of the sample mixture in the beaker pull the pluger back even more and shake the syringe ( mixture of sample and air)and then inject????. Really sorry I don't understand

4 Segment syringe technique.
Don't understand this technique at all. Sorry

Syringe
I have a 1ul syringe and I am trying to inject 0.1ul of sample.

Make-up gas flow
I set the make-up gass flow to the same as the carrier gas flow. I have this on all the time. I found this to make a difference to the peak shape. What does this do. I know the carrier is going through the column but the make-up???/


Experiment Results to date

I have done a few injections with the parameters I found to be good and the peaks are worse. So am going to change the injector and detector parameters and carrier gas flow.
I tried injecting 0.05ul of sample onto the column and the peaks were really sharp but the tailing was terrible which it is anyway.



Thanks everyone for the above suggestions and will go off and change the parameters again (because of column installation/ liner/ purge vent)

Michelle

A split ratio of 50:1 means that the split vent flow is 50 x the column flow. It's a ratio so it has no units. In your case the column flow is OK and estimating from your retention figures for unretained butane the flow is about 1.5 ml/min.

That means your split ratio is now 150/1.5 = 100:1, but that is also OK because you have been above, or on the upper limit of, your column capacity.

Any residual tailing you have is probably characteristic of your thin film stationary phase and not your new flow settings. The question is now - can it do the job that you wanted?

Dare I say it - it's an easy job for 2 metre packed column.

Hi,

Syringe

4 Segment syringe technique.
Don't understand this technique at all. Sorry

Syringe
I have a 1ul syringe and I am trying to inject 0.1ul of sample.

Make-up gas flow
I set the make-up gass flow to the same as the carrier gas flow. I have this on all the time. I found this to make a difference to the peak shape. What does this do. I know the carrier is going through the column but the make-up???/

Makeup

OK. We need to find out what the makeup flow shoule be for your detector. It should be in the manual. Because you GC can be used for both capillary ( 0.5 - 10 ml/min ) and packed columns ( 10 - 60 ml/min ) carrier gas flows from the column, it needs modifications to optimise it.

The most common modifications are changing the jet and gas flows. As the hydrogen/air ratio should be constant and is optimised for a midrange use. For example, many older detectors were setup for 15 - 30 mls/min of carrier, and the makeup gas is mixed with the carrier gas just before it hits the detector to ensure that sufficient flow is present to make peaks sharp.

If you have a low flow packed column or a large capillary at 10 ml/min you may want to add 10 - 20 ml/min of makeup gas. If you have a small capillary, you may want to add 20 - 30 mls/min of makeup. You need to find out what the detector requires from the manual. If you can not find it, increase makeup till you get nice sharp peaks, but do not exceed 30 ml/min.

Syringe.

A split-injection capillary GC without on-column concentration requires a near-instantaneous vaporisation of the sample and solvent to obtain really sharp peaks. Automatic injectors are fast, people aren't. What you need to do is get the syringe needle into the injector, add the sample, and withdraw the syringe as quickly as possible.

If your sample is in the needle, it will start vaporising out as soon as the needle passes through the septum. OK for some solvents and sample, and plunger-in-needle syringes have to do this, so really quick insertion and removal is usually necessary for sharp peaks and volatile solvents. If you have a plunger-in-needle syringe, you can't use the four-segment technique.

If the plunger is in the glass barrel of the syringe, there is the opportunity to keep the sample in the cooler syringe barrel whilst you carefully but quickly insert the syringe into the injector. Then you quickly press the plunger fully in and withdraw the syringe as soon as the plunger hits the bottom of the barrel. Some people prefer to leave the syringe in for a couple fo seconds once teh plunger has hit the bottton, counting "one" "two", and then withdraw the syringe. In that case some of the liquid in the needle will also boil out = larger injection. As long as you are consistent...

To produce a small injection of 0.1 ul, knowing that the injector is at a higher pressure, the sample has to be forced out, and air-gaps will compress as you push the plunger down. There will be some sample or solvent present that is equivalent to the volume of the needle.

So.
1. You first flush the syringe thoroughly with solvent. Then slowly push all the solvent out. Lift it out of the solvent and pull the plunger back and you will see some solvent in front of the plunger. that's the volume of the needle. You keep pulling the plunger back until the solvent/air front is at the 0.1 ul mark.
2. Without touching the plunger, insert into the sample and slowly pull the plunger back another 0.1ul. remove the needle from the sample and slowly pull the plunger back until the sample is all in the glass barrel, and the air is in front of it.
3. Inject as normal, but push the plunger quickly and withdraw syringe.

You will have plunger/solvent/air/sample/air/needle, hence four segment technique.

Hope this helps.

Bruce Hamilton

Hi,

What I meant was that I can't get pasting a quote to work. I'm sure it must be easy but I haven't figured it out yet. In the meantime I'll cut and paste.

"Empty syringe technique
Do you mean take up the 0.1ul of sample and then when the syringe is out of the sample mixture in the beaker pull the pluger back even more and shake the syringe ( mixture of sample and air)and then inject????. Really sorry I don't understand"

When you have taken up 0.1ul of sample and the syringe is out of the sample mixture just depress the plunger fully and briefly touch the tip of the needle on a piece of paper. You think that you have emptied the syringe but there is still a residual amount left in the needle. Then pull the plunger back to 0.1ul and inject as normal. It is just a way of injecting a very small amount.

Bruce's method is a far better one.

It would be worth contacting Shimadzu about the correct distance to insert the column into the injector - this distance is very important.

To MikeD - shall we write a paper "In praise of packed columns" ? It's long overdue.

Regards,

Ralph

Purge & Split Vent flows

Purge Vent Flow: I opened this valve and set the flow to around 2ml/min or 5sec/ 10ml

Split Vent flow: Measured this again. It is giving conflicting results. So I definetly set it this time to around 150ml/min or to be exact 3.6sec/10ml
If the method states that the split flow should be 1:50, does this mean 1ml in 50min or is it 50ml in 1min. I am getting confused with the units.
Should I adjust the split flow to 1:50 and will that allow me to operate at the higher temperature of 60C as stated in the method or should I just keep it at the 150ml/min

I should have commented on this. Firstly, always try to relate flows to mls/min. Using that unit will help you visualise flows, eg..
Carrier Gas In 50 - 200 mls/min
Septum purge 2 - 3 mls/min
Split Vent 30 - 200 mls/min
Column flow 1 - 5 mls/min
Make up gas 10 - 30 mls/min
FID Hydrogen 30 - 60 mls/min
FID Air 250 - 400 mls/min

In your case above, if you wanted the purge at 2 mls/min, then 10 mls should take 5 mins - not the 5 secs you suggest, which would give 120 mls/min. A high septum purge flow would begin to cause your split flow to seriously drop, as they are inter-related.

The carrier flow rate entering the splitter is controlled, and there are only three approved exits ( septum purge, split vent, column ), all the rest are unwanted leaks . The split flow is usually pressure controlled, so any flow rate changes will be compensated for.

As an rough example, if you feed in 155 ml/min of carrier at 200 kPa, and set the septum purge to 2 ml/min, the split vent to 150 ml/min, then the column is taking 3 ml/min, and you have a split ratio of 50:1.

As the column is a fixed resistance to flow, halving the pressure will halve the flow through the column to 1.5 ml/min, but the septum purge will only change very slightly, and the split vent will automatically increase to 151.5 ml/min. You would have to decrease the split flow to 75 ml/min to maintain the 50:1 split ratio.

Obviously, the split ratio ( (splitter flow rate + septum purge flow rate) / column flow rate ) has doubled. For most purposes, the septum purge flow of 2-3 ml/min can be ignored, but you can see that the easiest control of column flow rate is changing the carrier head pressure, and you then adjust the split vent flow to obtain the correct split ratio after setting a suitable head pressure to give optimum column velocity.

If you send too much of the carrier flow out the septum purge, the injection will be very variable, so septum purge is usually only 2-3 ml/min. Note that all of the above is a nasty generalisation, as temperature affects carrier gas viscosity which affects column resistance, which affects column flow, which affects split ratio etc etc..

Hope this helps.

Bruce Hamilton

Hi All,

Just to say thanks for all your suggestions so far. They have been really helpful and I have learned so much. I have learned far more here and I have applied it to my job and course.

I have trawled through all the posts and taken on board all suggestions and I thing that I have come to the conclusion that I will not achieve correct resolution with this column.

What column should I choose to achieve this separation?
I think that sticking with the 30m and maybe a 0.3um diameter??
What film thickness? 1.4um would 1.0um or 1.3um be ok??

Someone mentioned to me that I should set the detector and Injector temperatures to the following values respectively: 300C and 200C (min.) This is to ensure that all components are completely vaporised.

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

Thanks
Michelle

Hi All,

What column should I choose to achieve this separation?
I think that sticking with the 30m and maybe a 0.3um diameter??
What film thickness? 1.4um would 1.0um or 1.3um be ok??

Someone mentioned to me that I should set the detector and Injector temperatures to the following values respectively: 300C and 200C (min.) This is to ensure that all components are completely vaporised.

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

Thanks
Michelle

If your samples are fairly clean, then the 624 column will separate the compoents. It's worth noting that a polar column like 624 will bleed stationary pahse much more than your methyl silicone, so can't be taking to such high temperatures, and is more sensitive to oxygen.

The thicker the film, the more bleed, so if you have to heat the column to remove sample, then choose a thinner film thickness, and/or a less polar column. However, you will have to use a cooler oven temperature as the film thins. The suggested Det/Inj temp are OK, but you need to evaluate the optimum.

Ensure you follow all the instructions. Also, not all 624 are equal, I've encountered transposition of closely-eluting peaks by using columns from different suppliers. Most column suppliers will give specific details of solvent separation on their columns.

Bruce Hamilton

Michelle,

What columns do you have in your lab?

If all you want to do is to separate these two:

Use a trifluoropropyl methyl silicone capillary column and you will have over 2 carbon numbers separation. (830 and 1061) An OV-1301 or 1701 or a Carbowax capillary will also likely work.

OR

Use a 2ft 0.1% Carbowax 20M on Carbopack B packed column

or almost any porous polymer column of more than 2ft in length.

There are many ways to skin the proverbial cat.

best wishes,

Rod