Chromatography Forum

I am having some trouble using the USP method for calculating percent ethanol on the GC. Acetonitrile is used as an internal standard, so our standard vial contains 2% acetonitrile and 2% ethanol in water made weekly. I've noticed that when I make a new standard vial, all the peaks elute as expected where the ethanol and acetonitrile peaks are almost identical in height. After a day, sometimes a week, the acetonitrile peak will diminish and become substantially less than the ethanol peak which affects the calculated % ethanol in the sample vial. I was under the impression that acetonitrile doesn't readily react with ethanol or water. Where is the acetonitrile going? Do I just need to make new standard vials daily? Is the acetonitrile reacting with something over time? In order for the calculation to work, the acetonitrile peak needs to have the same area as the ethanol peak in the standard vial. I'd appreciate any suggestions as this has been extremely frustrating. Our lab is small and I'd like to avoid making new standards daily.

That's strange. You are correct, ACN doesn't react with water. I presume this is a static headspace analysis. Correct? Is it possible that your transfer lines are not as inert as you need them? How are the shapes of your peaks looking?

I am having some trouble using the USP method for calculating percent ethanol on the GC. Acetonitrile is used as an internal standard, so our standard vial contains 2% acetonitrile and 2% ethanol in water made weekly. I've noticed that when I make a new standard vial, all the peaks elute as expected where the ethanol and acetonitrile peaks are almost identical in height. After a day, sometimes a week, the acetonitrile peak will diminish and become substantially less than the ethanol peak which affects the calculated % ethanol in the sample vial. I was under the impression that acetonitrile doesn't readily react with ethanol or water. Where is the acetonitrile going? Do I just need to make new standard vials daily? Is the acetonitrile reacting with something over time? In order for the calculation to work, the acetonitrile peak needs to have the same area as the ethanol peak in the standard vial. I'd appreciate any suggestions as this has been extremely frustrating. Our lab is small and I'd like to avoid making new standards daily.

The fact that ACN peak hight decreases doesn't necessarily mean that it's area decreases.

Hello

Is your quantification method based on peak height or peak area?

Regards

Tomasz Kubowicz

That's strange. You are correct, ACN doesn't react with water. I presume this is a static headspace analysis. Correct? Is it possible that your transfer lines are not as inert as you need them? How are the shapes of your peaks looking?

We are not using a static headspace analysis. The samples we are testing are just water and ethanol. The transfer lines and carrier gas are certified GC grade and our peaks look excellent. Another piece of info: Our method asks that we run our standard (the one that's "degrading" after a few days - meaning the area of acetonitrile decreases over time and becomes less that the area of ethanol) and then we run our sample. Initially when I make the standard vial, the numbers come out great and both the standard vial peaks and the sample vial peaks look great. After a day or so, the sample peak areas will still come out as expected. The sample peaks look perfect and the acetonitrile peak area is equivalent to the ethanol peak area (we have to add acetonitrile to the sample as our internal standard that is then compared to the standard vial). However, the area of acetonitrile in the standard vial will begin to decrease which lowers the % ethanol numbers that are calculated.

I am having some trouble using the USP method for calculating percent ethanol on the GC. Acetonitrile is used as an internal standard, so our standard vial contains 2% acetonitrile and 2% ethanol in water made weekly. I've noticed that when I make a new standard vial, all the peaks elute as expected where the ethanol and acetonitrile peaks are almost identical in height. After a day, sometimes a week, the acetonitrile peak will diminish and become substantially less than the ethanol peak which affects the calculated % ethanol in the sample vial. I was under the impression that acetonitrile doesn't readily react with ethanol or water. Where is the acetonitrile going? Do I just need to make new standard vials daily? Is the acetonitrile reacting with something over time? In order for the calculation to work, the acetonitrile peak needs to have the same area as the ethanol peak in the standard vial. I'd appreciate any suggestions as this has been extremely frustrating. Our lab is small and I'd like to avoid making new standards daily.

The fact that ACN peak hight decreases doesn't necessarily mean that it's area decreases.

Sorry, we use area not height. The area decreases. The height decreases also in this case, but we do not use height in our calculation.

Hello

Is your quantification method based on peak height or peak area?

Regards

Tomasz Kubowicz

Our quantification method is based on peak area. We take the area of ethanol from the sample (Esample) divided by the area of acetonitrile from the sample (Asample) all divided by the area of ethanol from the standard (Estandard) divided by the area of acetonitrile from the standard (Astandard).

(Esample/Asample)/(Estandard/Astandard)

just make new vial every day, acetonitril is very volatile.

This sounds like USP <611>, for alcohol content. Since we manufacture products containing 60-70% ethanol, we had to validate this procedure for our own finished products, as the USP procedure is only for alcohol-water solutions.

1. The aqueous solutions of ethanol-ACN and ACN internal standard + sample are injected as liquid, 0.2 to 0.5 microliters.

2. The USP <611> column detailed (a "624" column) is terribly sucky for aqueous injections, the ethanol peak tails a fair amount. Sometimes we fail system suitability for tailing.

3. We've always made up our 2% ethanol standard in water fresh every day. The ethanol-ACN mixture is made from that and our 2% ACN. If that 2% ACN is well-mixed and then used both to make the Rf standard mix and for adding to aqueous samples, then the 2% ACN does not need to be made fresh every day.

4. We have plans to ditch the 624 column for a PEG-type column which can handle aqueous injections better, switch to hydrogen carrier gas/nitrogen make up gas, and do a revalidation in 2015. The AOAC procedure we used prior to this used injections from aqueous on a PEG-type column, and never once did we have a tailing issue. And the first column for this assay (18 years old now) still works fine.

Seaweed may well be an ideal plant to turn into biofuel. It grows in much of the two thirds of the planet that is underwater, so it wouldn't crowd out food crops the way corn for ethanol does. Because it draws its own nutrients and water from the sea, it requires no fertilizer or irrigation.

http://www.thegreenbook.com/products/packings/

Seaweed may well be an ideal plant to turn into biofuel. It grows in much of the two thirds of the planet that is underwater, so it wouldn't crowd out food crops the way corn for ethanol does. Because it draws its own nutrients and water from the sea, it requires no fertilizer or irrigation.

http://www.thegreenbook.com/products/packings/

This "may" be "off-topic"....

I would think a 2m at least retention gap would be a good idea to protect the analytical column with a liquid water injection from the nonvolatiles. Also a column rinse kit would be useful as well.

I use a wax for ethanol but I noticed it doesn't get much resolution between isopropanol and ethanol which I was considering using IPA as an internal standard.

For ethanol I have used a blood Alcohol column as well as a FFAP (wich is a Wax type). Both work fine for most solvents... But go with a relative thick film for the low boilers.

- Karen

I would think a 2m at least retention gap would be a good idea to protect the analytical column with a liquid water injection from the nonvolatiles. Also a column rinse kit would be useful as well.

I use a wax for ethanol but I noticed it doesn't get much resolution between isopropanol and ethanol which I was considering using IPA as an internal standard.

Typically n-propyl alcohol (AOAC) or ACN (USP) are usefull internal standatrds for ethanol.

This one intrigued me and I have an instrument that needs a little bit of a workout. I made a water solution of 2% EtOH and 2% ACN and analyzed it in triplicate at time zero as well as every day for nearly a week. During storage, the water/EtOH/ACN mixture sat on the bench, at RT, in a stoppered 100 mL volumetric flask.

My method is a heated-headspace SPME method (no water injection). Here are the data:

https://flic.kr/p/pzALG6

Something was out of the ordinary on day 0 as the precision was the worst on that day and the absolute response for ethanol was the greatest in that set. After day 0, I got essentially the same answer and approximately the same precision each day. The last 2 columns take the data as it is and calculate the concentration of EtOH in the sample using the indicated response factor - one uses the average RF from all of the data and the other uses the average RF that excludes the day 0 data).

It appears that if you're losing ACN, it must be related to how the sample is introduced into the chromatograph. In the water standard, it appears to be fairly stable. I can't really explain why day 0 is so imprecise in my data. I mixed the contents of the flask very hard with shaking and swirling. As far as the actual analysis, everything was done the same way, every day.

Sampling:

2 mL of solution in a 22 mL headspace vial
SPME with a 100% PDMS fiber
15 minute preheat to 60 °C (in a dri-bath heater with the block to accomodate the headspace vials)
30 minute extraction of the headspace at 60 °C

Instrument: HP 5890 Series II
Column: Zebron Wax, 30 m x 0.53 mm x 1.0 µm (Phenomenex)
Inlet: 230 °C, helium carrier gas at 2.5 psig, 5:1 split, SPME inlet liner
Oven: 40 °C for 2 min., 40-230 °C at 10 °C/min., 230 °C for 14 min.
Detector: FID at 230 °C, 30:300:25 H2:Air:helium makeup gas