Quantitative Analysis
In chromatography there are two types of analysis ‘qualitative analysis’ which identifies the solute or solutes present in a mixture and ‘quantitative analysis’ which determines how much of a substance or substances is present in a mixture. Qualitative data is obtained from retention measurements; quantitative data is obtained from peak height or peak area measurements. Quantitative analysis makes certain demands on the chromatographic apparatus, particularly the injection system and the detector. In the first instance, a truly representative sample must be placed on the column by the injection system and secondly, the detector must have a linear response that is known and defined by its response index. For accurate work the response index of the detector should lie between 0.97 and 1.03 over the concentration range used. Providing the separation is highly reproducible, peak heights can be used as a relative measurement of the quantity of material present. To obtain a value proportional to the mass of solute present, a ‘response factor’ must be used for each substance determined and these response factors are obtained by prior calibration. Alternatively peak areas can be used for quantitative assessment and peak area measurements are claimed to be more precise. Peak area measurement may also be necessary if peak profiles are not close to Gaussian in shape and significantly distorted. Almost all quantitative measurements are obtained using an internal or external standard chosen so that it is eluted discretely and well separated from other components of the mixture. Quantitative analysis is probably the major application of chromatography techniques.
Author: RPW Scott
Book:Gas Chromatography
Section:YES Applications Food-and-Beverage-Products
bacterial action, aging, rancidity or
decomposition. In addition, tests that identify the area or country in which
the food was processed or grown may also be needed. The source of many plants
(herbs and spices) can often be identified from the peak pattern of the
chromatograms obtained directly from head space analysis. Similarly, unique
qualitative and quantitative patterns from a GC analysis will often help
identify the source of many alcoholic beverages.
Unfortunately,
food analysis involves the separation and identification of very complex
mixtures and the difficulties are compounded by the fact that the components
are present at very low concentrations. Thus, gas chromatography is the ideal
(if not only) technique that can be used successfully in food and beverage
assays and tests.
The potential
carcinogenity of the aromatic hydrocarbons make their separation and analysis
extremely important in environmental testing. However, the aromatics can pose
YES Applications Food-and-Beverage-Products
Author: RPW Scott
Book:Principles and Practice of Chromatography
Section:Principles Applications Gas-Chromatography Gasoline
for the separation of hydrocarbons (e.g.
OV101, which is also a polyalkyl-siloxane, is widely used in packed columns).
The flow velocity of 20 cm/sec., appears to have been taken from the ratio of
the column length to the dead time. Thus, due to the pressure correction the
actual effective linear velocity would be much less than that (see
Dispersion in Chromatography Columns ). Helium was used as the carrier gas
which was necessary to realize the high efficiencies with reasonable analysis
times. The FID detector provided the necessary to wide quantitative dynamic
range. The column temperature was held at 35˚C for 15 min. to effect the
separation of the low boiling, low molecular weight hydrocarbons, the
temperature was then increased to 200˚C at 2˚C/min. and finally held
at 200˚C for 5 min. to ensure the complete elution of the higher boiling
components.
An excellent
separation is obtained giving clearly separated peaks for the marker
Principles Applications Gas-Chromatography Gasoline
Author: RPW Scott
Book:Gas Chromatography
Section:YES Quantitative-Analysis
Quantitative Analysis
There are
three important stages in a GC analysis,
1. The
preparation of the sample.
2. The
development of the separation and the production of the chromatogram
3. The
processing of the data and the presentation of the results.
Each stage is
equally important and if not carried out correctly the results will be neither
precise nor accurate. Sample preparation can be very simple involving no more that
diluting a known weight of
YES Quantitative-Analysis
Author: RPW Scott
Book:Extra Column Dispersion
Section:EC-Dispersion Response-Time
nbsp;
The
quantitative evaluation of seven duplicate analyses is shown in Table 5.
Table 5.
Reproducibility of the Quantitative Analysis by Peak Area Normalization
of a Five Component Synthetic Mixture (Isocratic High Speed Separation)
Compound
Normalized Peak Area
Mean
Mean s
s %
p-Xylene
9.8
9.8
9.6
9.8
9.8
9.6
9.7
0.10
1.1
Anisole
EC-Dispersion Response-Time
Author: RPW Scott
Book:Gas Chromatography
Section:YES Applications Free-Fatty-Acids-from-Milk
to automation either
appropriately designed hard-wired equipment or by the use of a laboratory
robot. The hard wired device is generally inflexible, the laboratory robot, on
the other hand, can be programmed to carry out many different types of
analysis.
The separation
itself has some interesting properties. Free acids are very readily adsorbed
onto active sites on the support which can result in very asymmetric peaks and,
as a result of the strong adsorption, significant quantitative losses can
occur. In the above example, the effect of the adsorptive sites on the support
is reduced by blocking them with phosphoric acid. Phosphoric acid is very
involatile and thus can tolerate the high temperature and although it is active
enough to block the adsorption sites it is not active enough to cause sample
decomposition. It is seen that the peaks exhibit excellent symmetry for free acids.
Teraphthalic acid has also been used for this purpose to deactivate the
YES Applications Free-Fatty-Acids-from-Milk
Author: RPW Scott
Book:Liquid Chromatography
Section:HPLC Refractive-Index
detector is often a 'choice of last resort' and is selected for those applications
where, for one reason or another, all other detectors are inappropriate or
impractical. However, the detector has one particular and
unique area of application and that is in the separation and analysis of
polymers. For those polymers that contain more than ten monomer units, the
refractive index is directly proportional to the concentration of the polymer
and is practically independent of the molecular weight. A quantitative analysis
of a polymer mixture can, therefore, be obtained by the simple normalization of
the peak areas in the chromatogram (there being no need for the use of
individual response factors). Some typical specifications for the refractive
index detector are as follows:-
Typical
Specifications for a Refractive Index Detector
Sensitivity (benzene)
1x 10-6 g/ml
Linear Dynamic Range
1 x 10-6 to 1 x 10-4 g/ml
Response Index
HPLC Refractive-Index