Question

How to read a gas chromatography graph. How to find retention time and integration of each peak? How to find the major product from the graph

Answer 1

to Read a Chromatogram?

Over the years chromatography has gained an enviable position in analytical laboratories involving separation and quantification of organic compound mixtures. However, a chromatogram is not a display of results in concentration units but rather a graphical display in real time of peaks generated as the separated components pass through the detector.

The chromatogram makes little sense to the layman as the peaks provide no information on the identity of the mixture components nor any information on the amount present.

First of all it is necessary to understand what a chromatogram depicts. The chromatogram is a two-dimensional plot with the ordinate axis giving concentration in terms of detector response and the abscissa represents the time. The detector gives response as a peak whose height should be ideally dependent on concentration of the particular component.

Retention Time (tr)

However, due to analysis conditions peaks may deviate from ideal shape and peak height can no longer be a true measure of the concentration and instead the area under the peak is considered as a measure of component concentration.

Each peak represents a component present in the sample. Retention time is time interval between sample injection and the maximum of the peak. It is characteristic of the identity of the component under the operating conditions. Identity of the component can be confirmed by making injections of reference material under the same operational conditions. The matching of retention time of reference material and the component peak confirms the identity of the unknown sample component.

Now let us consider a sample which contains more than one sample component. Likewise each component will be eluted at different retention times depending upon solute – stationary phase interactions and mobile phase flow characteristics.

Calculation of results

From the area measurements using simple arithmetic it is simple to calculate the concentration of each component as a percent of the total.

%A =

Real Chromatogram

Let us now look at the actual chromatogram printout of HPLC separation of a mixture of vitamins A and E in a food matrix and see what the chromatogram represents

Actual Chromatogram

The ordinate is in units of volts and abscissa in minutes. The signals are recorded at a wavelength of 284 nm using a UV detector.

Retention time of each peak is marked above the peak and in the tabulated data below the chromatogram details of the retention time, area (as digital units), peak area%, height and height %. You can observe that due to non-ideal shape of peaks percentage area is different from percentage height for each component so area measurement is a more reliable measure of concentration.

You have been introduced to simple concepts on how to read a chromatogram. Please let us have your comments on the article and if you found it useful.

Calculating the Retention Time

The retention time is calculated according to the following equation: What is the retention time for Peaks A and B below if the chart speed was 2 cm/s? ANSWER = 7.5/2= 3.75 s The integrated intensity of a signal in a 1H NMR spectrum (does not apply to 13C NMR) gives a ratiofor the number of hydrogens that give rise to the signal, thereby helping calculate the total number of hydrogens present in a sample.NMR machines can be used to measure signal intensity, a plot of which is sometimes automatically displayed above the regular spectrum. To show these integrations, a recorder pen marks a vertical line with a length that is proportional to the integrated area under a signal (sometimes referred to as a peak)-- a value that is proportional to the number of hydrogens that are accountable for the signal. The pen then moves horizontally until another signal is reached, at which point, another vertical marking is made. We can manually measure the lengths by which the horizontal line is displaced at each peak to attain a ratio of hydrogens from the various signals. We can use this technique to figure out the hydrogen ratio when the number of hydrogens responsible for each signal is not written directly above the peak (look in the links section for an animation on how to manually find the ratio of hydrogens as described here). Now that we’ve seen how the signal intensity is directly proportionate to the number of hydrogens that give rise to that signal, it makes sense to conclude that the more hydrogens of one kind there are in a molecule (equivalent hydrogens, so in the same chemical environment), the more intense the corresponding NMR signal will be. Here's a model that may help clear up some of the uncertainties. Study tips When trying to build a structure for an unknown molecule from NMR data, find a common divisor that reduces the integrated areas of all signals to small whole numbers when the exact number of hydrogens responsible of each peak is NOT given. If, for example, the total number of hydrogens you count from your NMR spectrum is 5, but you know that there are supposed to be 10 hydrogens in the molecular formula, then it is a sure sign that the your molecule is symmetrical. You only find half the hydrogens in your NMR spectrum because the symmetry places hydrogens in the same chemical environment-- giving rise to the same peaks. (Again, the spectrum simply gives you a ratio.) A peak with 3 hydrogens commonly belongs to a -CH3 group.