Improving linearity

[Mac2]

Hi,
I'm developing a HS-GC method on an Agilent system however I'm having problems with linearity. Does anyone have any ideas on how to improve linearity so to achieve a result "not significantly different from zero"?
I'm currently running levels from 0.2 ppm to 5 ppm. Does it help to run levels equally separated or run more injections at each level??

[Stunt]

Equally separated curve points are the way to go, although this usually aids more in analysis than calibration. Start at 0.2 and double or triple your concentrations. Eg: 0.2, 0.6, .18 etc. If you can't achieve a passing linear curve, you could try average RF or quadratic as well.

[chromatographer1]

Mac2

Perhaps your instrument is trying to tell you something.

Look at your parameters and change them to see if you are doing something inappropriate.

Headspace is the most accurate reproducible technique I have ever used (except for weighing samples).

If you care to describe what EXACTLY you are doing I would be glad to comment.

Rod

[Mac2]

Thanks for the replies.

Rod,
Basically I am adding known concentrations of Toluene into 5 mL of sample (20 mL vials) and measuring the Toluene peak areas I'm getting back after analysis. My split ratio is 0.1:1 so as to maximise the peak areas. Sample injection is 1 mL, incubation time is 1 hour at 85 degrees, Loop and transfer line temperature are both 110 degrees. Pressurisation time is 0.4 mins, injection time 0.5 mins, loop fill time 0.08 mins and loop equilibration time 0.2 mins.
My RSD for 6 injs tends to be 5-10%.

[Mac2]

Stunt,
What do mean by try average RF or quadratic as well???

[chromatographer1]

Let me dig some more facts out of you. (details DO matter)

How much toluene do you add to each vial holding 5mL of WHAT SAMPLE?

These little BITS of information are more important than you realize.

You only fill the loop for 0.08 min? Whatcha hurry?

Fill the loop until atmospheric equilibrium is achieved (watch your sample bubble out the sample vent line until it stops)

5-10% RSD values for toluene are AWFUL.

1% is middle of the road.

In other words: your method is a bit crusty and needs a little polishing.

best wishes,

Rod

[Mac2]

5 microlitres of a 1000 ppm Toluene solution (diluted with dimethylacetamide) is added to 5 mL of our product to give a 1 ppm standard.
What would be a typical loop fill time?

[chromatographer1]

ppm nomenclature is so very ambiguous ! wt/v, wt/wt, v/v, etc. One should clear scientific nomenclature. SORRY to be so snotty, but I know many will agree with me about this. So I am guessing, correct me if I misjudge:

5 microliters of a 1000 ppm solution of toluene in DMAc, or 5mcL of 1mL of toluene per liter of DMAc, or 5mcL of 1mcL per mL of toluene in DMAc, or 5 mcL of a a 1 nL toluene per microliter of DMAc.

That means you are adding 5nL of Toluene per 5mL of unknown sample = 1 ppm v/v

OR

4.32ng per 5mL of unkown sample

Am I right so far?

If the sample is aqueous then 10 min is enough heating, if non-polar, then may be 15 min.

I would expect at least a less than 3% RSD. A simple vial of water can test your sample vent line for bubbles. When your sample stops bubbling through or if your sample loop has been flushed 3-5 times, then that is enough time, but the sample flow MUST be stopped and the line come to equilibration before you inject.

best wishes,

Rod

[Johnny Rod]

Are you using an internal standard? What flows are you using for HS and GC?

[Mac2]

Yes Rod that is correct, 1 ppm v/v.
Any ideas on improving linearity specifically? I have a lot more levels at lower concentrations, should I be using as many levels at higher concentrations to improve values? Do more injections at each level help?

I am using a constant flow of 5 mL/min resulting in a split flow of 0.5 mL/min. No internal standard is used.

[chromatographer1]

Linearity issues have many causes. Your linearity should be at least 0.9995, otherwise something is not optimized.

1. Your heating - mixing protocol may not be optimized. This is the most likely problem. Heating more than 30min should NEVER be necessary unless you are using too large a sample. Larger samples do not always mean bigger peaks. Yeah, I was surprised at this too 20 years ago when I did my research.

If you are splitting your sample this can cause issues. Personally I never liked or used splitting headspace. If you go to all the trouble to use big samples (5mL is not small) why then deliberately throw away 50% OR MORE of the sample ?

Toluene is easily focused and you can probably use normal capillary pressures for injection, but at least use splitless injection (10 secs or more before split vent opens). Review how PE headspace analyzers work differently from Agilent's. Many believe PE units are superior to their competition. All can be made to work at great effectiveness if operated correctly.

I preferred increasing the head pressure to 20 psi or more and decreasing column flow by restrictor tubing to improve focusing of the sample plug. I was able to achieve linear results from 0.1ng per vial to 1000 micrograms per vial for toluene (R=0.99990) You can read my paper in Analytical Chemistry on trace analysis with HS (June 1995). My samples in this paper were 0.5ng to 1000ng per vial. See the effect of vial size on analyte peak height/area.

2. Vial seals, cleanliness of transfer lines, and installation of head of column in the injector are other problems which can cause linearity issues.

3. Over pressuring of the vial after equilibration, this is before injection, or not allowing the headspace to equilibrate after pressuring the vial before injection can be another cause of linearity issues.

The easiest way to determine optimum parameters is to use more concentrated samples with a larger toluene peak to determine if you get better RSD values or just more repetition of the errors found at the trace levels.

My comments would be: use 1mL or less in 6mL vials rather than 5mL in 20 mL vials.

Use direct injection of 0.25-0.5mL loop instead of splitting the sample. At least use splitless injection instead of a simple split injection.

Use a megabore column attached to a 5 meter piece of 0.25mm ID column to improve focusing of the sample plug. Use 5mL/min to 20mL/min column flow rates.

Good luck,

Rod

[Mac2]

Thanks a mil for that Rod. I'll definitely give some of those suggestions a go!

[chromatographer1]

I forgot to mention, when heating for extended periods, any inconsistent sealing issues will surface as high RSD values. Another reason to never heat longer than necessary.

Rod

[Johnny Rod]

I used to use a HS-GC setup with split inlet. One of the things people would do is set the HS carrier flow at, say 100ml/min when the total inlet flow was, say 50ml/min, so most of the sample ended up disappearing out the split vent and the GC had no chance of controlling the inlet as it was being fed too much gas anyway. As Rod says, injecting all of your sample would be a better bet!

[Mac2]

Guys,
Agilent do not recommend using splitless injection for a number of reasons