Structural Biochemistry/Chromatography/Gas
Gas Chromatography is common type of chromatography which is used to analyze or separate volatile components of a mixture. This technique helps us to test the purity of a particular substance or separate different components of a structure. Basically, the mechanism of this technique is carried out by injecting syringe needle which contains a small amount of sample into the hot injector port of gas chromatography. The injector is set to the temp that is higher than the boiling points of the components so that the components will be evaporated into gas phase inside the injector. The carrier gas (normally is Helium) then pushes the gaseous components into gas chromatography column. The separation of components occurs here, form partition between mobile phase (carrier gas) and stationary phase (boiling liquid). More interestingly, gas chromatography column showed what’s inside, the maximum temperature along with the length and diameter due to the presence of metal identification tag on the column. Additionally, the column temperature is raised by the presence of heating element. The detector inside the gas chromatography will recognized the differences in partition between mobile and stationary phases. The molecules reach the detector, hopefully, at different intervals depending on their partition. The number of molecules that regenerate the signal is proportional to the area of the peaks.
Although gas chromatography has many uses, GC does have certain limitations. It is useful only for the analysis of small amounts of compounds that have vapor pressures high enough to allow them to pass through a GC column, and, like TLC, gas-liquid chromatography doesn't identify compounds unless known standards are available. Coupling GC with a mass spectrometer combines the superb separation capabilities of GC with the superior ID methods of mass spectrometry. GC can also be combined with IR spectroscopy. IR can help to identify that a reaction has gone to completion. If the functional groups of the product are depicted in the IR, then we can be sure that the reaction has gone to completion. This can also be depicted in the GC analysis. The presence of peaks that do not correlate with the standards may be due to an incomplete reaction or impurities in the sample.
The basic parts of a GC machine are as follows:
- Source of high- pressure pure carrier gas
- Flow controller
- Heated injection port
- Column and column oven
- Detector
- Recording device
A small hypodermic syringe is used to inject the sample through a sealed rubber septum or gasket into the stream of carrier gas in the heated injection port. the sample vaporizes immediately and the carrier gas sweeps it into the column. The column is enclosed in an oven whose temperature can be regulated. After the sample's components are separated by the column, they can pass into a detector, where they produce electronic signals that can be amplified and recorded.
The steps need to be followed to use Gas Chromatography:--Cherryblossom06 (discuss • contribs) 06:02, 22 November 2012 (UTC)
1. Wash syringe with acetone by filling it completely and pushing it out into a waste paper towel.
~Possible errors that can occur during Gas Chromatograpy can be due to the improper rinsing of the syringe. The syringe should be rinsed twice with acetone and once or twice with the sample. If improper rinsing ensues, unknown peaks can occur and alter our analysis of the sample. This error can be easily avoided. About 1 micro liter of sample is needed.
2. Pull some sample into the syringe. Air bubbles should be removed by quickly moving the plunger up and down while in the sample.
3. Turn on chart recorder, adjust chart speed in cm/min, set baseline by using zero so that the baseline is 1 cm from bottom of chart paper ( set 0), turn on the chart.
4. Inject sample into either column A or column B and push the needle completely into the injector till we can’t see the needle, then we pull the syringe out of the port.
5. Mark the initial injecting time on the chart. ~The sample should be injected at exactly the same time as the 'start' button is pressed. Otherwise, take note of how long after injection recording started. If the sample is not injected at the exact time the button is pressed, retention times will be off in the calculations.
6. Clean the syringe immediately.The syringe should be rinsed with acetone before injecting a different sample. Rinse before any other sample is injected and after every sample.
7. Record current (in milliamperes), temperature (in Celsius).
Notes on Injection:
1. The injection site, the silver disk, is very hot.
2. The needle will pass a rubber septum so there will be some resistance. Some machines have a metal plate near the septum, so if there feels like metal resistance, the needle should be pulled out and tried again. The needle should be completely inserted into the injection point if done correctly.
3. Quick injection is needed for good results.
4. Take out the needle immediately after injection.