Chromatography Forum

I'm a recent GC convert and have some newbie questions regarding standard curves for quantitation. My setup is a Shimadzu 14A, Restek ShinCarbon ST 100/120 column, TCD detector, N2 carrier gas, and our injection size is 500ul. We are following headspace gas levels of bacterial cultures and would like to quantify H2 (.01-100.00%), CO2 (0.0-5.0%), and O2 (0.0-20.0%). At first, quantifying H2 will be the most important. My questions are:

1. Any idea on the Shimadzu TCD linearity over these ranges? I have yet to find a good reference for this and there is little GC expertise around our lab.

2. Any suggestions for gas mixtures for standard curves (remember, we're most worried about H2)? I'm assuming this is highly dependent on linearity.



Thanks!
Dave Savage

Dave,

Trying to measure CO2 and O2 using Nitrogen as carrier is not performed with much success. Examine the thermal conductivities. It is optimal for Hydrogen however, and almost necessary. Argon is an alternative.

The design of the detector is critical for linearity with TCD. I have no information on your GC to share with you.

Your stds should be encompassing your entire measurement range. The choices of 10%, 50%, and 120% of maximum range are a very minimum. More points would be better. How many can you afford?

You also need to determine the limit of accurate and repeatable detection. This will require additional work.

You may find a porous polymer to be a better choice for this analysis. A 10 foot Hayesep or Porapak Q 100/120 mesh might be a better column for your work. (Since no N2/O2,C1 separation is necessary)

Good luck,

Rod

The hydrogen calibration curve will not be linear with most samples, unless a special carrier gas blend is used. This has been covered here before, and is discussed in most gas analysis books.

IIRC, the out-of-print book, THE ANALYSIS OF GASES BY CHROMATOGRAPHY by C. J. Cowper and A. J. Derose. Hardbound, ISBN: 0-08-024027-5, 159 pages, publication date: 1983 Imprint: PERGAMON, has a deteiled discussion, and is full of information very good for most gas analysis techniques except those iusing modern capillary columns, such as PLOT types, which Chrompack used to have alot of information about.

I'm not sure that you will get 100% hydrogen, and you should initially calibrate for what the literature indicates you should expect, and choose standards in those regions.

If necessary, you can make up quick and dirty standards for trials using a large gas-tight syringe and using a cheap 3-way luer-lock plastic valve ( used in medical drip lines etc ) to add components from smaller syringes.

Bruce Hamilton

Dslav,

I just finished an install in a similar application. The O2 and CO2 will be well resolved but don't forget to take into account that water will eventually be coming off. As per Rod, the sensitivity towards the CO2 and O2 is probably going to be very poor. You might consider, if you have the time, switching from nitrogen for hydrogen to helium for CO2 and O2.

The application we did was a TCD in parallel to an HID with helium carrier (split at the column outlet), the HID for hydrogen (although saw the others) and the TCD for O2, N2, CO, CH4 and CO2 on the 2 meter ShinCarb. 50C for about 6 minutes then 15 C to about 150C. Linearity on either detector in this configuration has not yet been established.

Dslav,
I am running similar experiments: Detecting hydrogen from the headspace of the bioreactor.

I am using a mole-sieve column, with argon as carrier gas.
I pretty much get a linear curve for increasing hydrogen concentrations.

However, i can only separate h2,o2,n2,ch4, with my column, and not suitable for co2 measurement.

Regards,
Ramana

Thanks to all for excellent and informed replies.

I just thought I would post an update for anyone else with similar needs and/or equipment.

As I discussed above, we are taking headspace samples from biological cultures and mainly interested in hydrogen detection. We have many different types of cultures and were shooting for a wide range of hydrogen detection and quantitation, something lilke .1% to 100%. For the time being we have decided to worry less about other gases and focus on hydrogen.

Per the suggestions, we used pure hydrogen to mix our own standards. The detector has two regimes, .1-10% and 10-50%, which are linear. Above 50 it's highly non-linear and we have yet to explore much below .1%.

Thanks again for the help!


-dave

Dave,
How do you prepare hydrogen gas standards? Sample Bags??
Ramana

dslav,

I found the same thing with hydrogen. I used the point to point calibration method and ended up using 3 points for a satifactory curve. 0-5, 5-30,30-100%. Additional points between 30 and 50 and 0 and 1 would have been better. I also found that the peak polarity changed over 5%. Oh, I'm use helium as a carrier.

Good luck