Chemistry 227 – Fall 2009
Lab 6: Chromatography
Chromatography is one of the most widely used methods for chemical separation.
lab, you will learn about the principles of chromatography, primarily through experiments with
gas chromatography (GC), but you will also have a brief experience with high performance
liquid chromatography (HPLC).
You will explore the role of stationary phase in separation and
the factors affecting resolution, use the Kovats index to aid in qualitative identification of
compounds, and make a quantitative determination on a sample by standard addition.
addition, you will see how to manipulate mobile phase and detection to improve HPLC
Use care when handling organic compounds in this lab.
Do not leave containers open, do
not take large quantities of any solvent or mixture, and use care not to spill substances on
yourself, on any equipment, or on the computer.
Use caution when you handle the syringe and
make injections; the needle is sharp and the injection and exit ports of the GC are hot.
of excess reagents in the appropriate container.
Be sure that vials containing samples and
solvents are kept tightly closed when not in use.
The gas chromatographs should be ready to operate when you arrive in the lab, except for
final adjustments of the column flow rate and the detector bridge current.
About 4 hours are
required for the GCs to stabilize at the operating temperature, so don’t adjust the temperature
settings without first consulting a lab instructor.
Begin this experiment by locating the various
controls and ports on the GC; these are the current/temperature meter, meter selector switch,
injection ports for columns A and B, flow adjustment controls for columns A and B, exit ports on
the left side of instrument, detector power switch, detector polarity switch, detector bridge
current control, attenuator selector switch, zero adjustment control, and temperature controls for
the injector ports, detector, and columns.
Do not adjust any controls until the instructor or a lab
assistant has reviewed the GC operation procedures with you and answered your questions.
However, you may use the meter selector switch to check temperatures and the detector bridge
The following control settings are the target for this experiment:
column temperature — 110 ºC
detector temperature — 130 ºC
injector temperature — 130–135 ºC
detector bridge current — set to its maximum (should be at least 225 mA)
Adjust the attenuator so that the peaks for eluting compounds are on-scale (i.e., between ±50
mV; peaks are larger when the attenuator setting is smaller).
Start with the attenuation settings,
injection volumes, and flow rates suggested in Table I below, and then make adjustments as
necessary. When the polarity switch is in the + position, positive peaks will be seen for injections
on column A, and negative peaks, for injections on column B.
The detector output zero