Qualitative & quantitative analysis

We typically think of GC and LC as quantitative tools. In general, chromatography is a blind method. It indicates the presence of a substance but not what it is. Even so, qualitative data can be obtained even with non-discriminating detectors.

Qualitative analysis
Retention data can be used for some qualitative work. The tR is characteristic of a substance, compared to a standard. To be useful, some problems must be addressed. Reproducibility of absolute retention data depends on several experimental conditions. Is tR, vR, vR or tR best to use?

Retention time - tR - time elapsed from point of injection to maximum of peak. Adjusted tR - tR - time from maximum of unretained peak to maximum of eluent. Hold up time - tM - time required for mobile phase to traverse the column.

Retention volumes
If the flowrate (Fc) is constant and known then:

tR

Retention volume = Adjusted VR Hold up volume = =

VR

= tR Fc

tm

VR = tR Fc Vm = tM Fc

tR

Retention relationships
Retention volume or time may be used for identification. For a homologous series, VR can be accurately determined by: ln Vn = a + bn where Vn and n a, b Vn = = = adjusted retention volume carbon number fit parameters

Retention relationships
To determine an unknown carbon number:

x = n1 + ]n 2 - n1g

ln V x - ln Vn ln Vn - ln Vn
2

1 1

where n 2 > x > n 2

This can only be used for straight chain compounds and the unknown must fall between n1 and n2. Expressed as integer.

= Vn - Vm

Absolute retention index

To evaluate species that are not members of a homologous, we calculate an index value like it was a paraffin. The value does not need to be a whole number.

Kovats retention index

A modification of the absolute index where:

Ik = 100IP Ik = 100n1 + 100]n 2 - n1g ln V x - ln Vn ln Vn - ln Vn

2 1 1

ln V x - ln Vn IP = n1 + (n 2 - n1) ln Vn - ln Vn where n 2 > x > n1

2

1 1

This index has been determined at different temperatures for a large number of compounds. Tables are also available. The value can be used to compare related separations.

n2 and n1 are reference paraffins.

Other

homologous series could be use but paraffins are the norm.

Relative retention data

One practical approach for your own data is the use of relative retention.
' ' tR VR k unk ri = ' = ' unk = unk k std tR std VR std

Relative retention data

To be useful Standard should be a part of the sample or added to it - internal standard. It should be something that: Elutes near center of an analysis. Uses a sample size. Values will remain pretty constant between runs - may vary with a new column. Only for isothermal/isocratic conditions.

This is a common approach. It only requires a single standard. If the standard is the last peak to elute then ri is called the retention index.

Retention time, tR
Retention time data is adequate for simple assays like process quality control. ! You already know what is there. ! There are only a few components in the sample (or only a few of interest).

Other methods
Retention plots Retention values of materials belonging to a homologous series can usually be related to physical characteristics. In many cases, a semi-log plot of tR vs. carbon number will give a linear relationship for earlier members of a series. This can be used to pick out potential series members.

If a true unknown is observed, you cant do much more than note its presence!

Other methods
Solvent 1
Peak shifting in liquid chromatography Unlike with GC, LC allows for changing the nature of the mobile phase. Altering the solvent can be used to change the elution times and orders. This, with the addition of standards can often give a good match.

Peak shifting
Solvent 2

Other methods
Post-column methods ! Post-column collection and analysis using a separate qualitative tool. ! Quant/qual detector. Couple the GC or LC to a discriminating detector.

Quantitative analysis
GC or LC + FTIR, MS, AA UV/Vis, Emission
All chromatographic detectors produce a signal that drives a meter, recorder, integrator or A/D converter. While the detectors used for GC and LC are not the same, quantitative methods are identical. Each detector will produce a response/unit concentration. This is substance dependent so standards must always be used.

Peaks
Each quantitative method assumes that you have one or more reasonably resolved peaks. You must be able to find the beginning and end of each peak as well as its maximum.

In some cases, you can assume that peak height is proportional to concentration. Advantages Simplicity Rapid calculations Disadvantages Height is more variable than area Typically used only with capillary columns

Peak height

Peak area
This is the major approach for establishing a relationship between peaks and concentration.

Peak area
If the peak is approximately Gaussian, how do we accurately measure its area? Manual Automated Cut & Weigh Integrating recorder Planimeter Digital integrators Triangulation Computer systems Today, stand alone digital integrators and computer systems are the norm. Still good to review earlier approaches.

area ! concentration
Area is determined from a large number of measurements and detectors usually have very large dynamic ranges. This results in a very reliable measurement.

Cut and weigh

With this approach, each peak is cut from the recording paper and weighted. Weight is then considered proportional to area.

Planimeter
A device used to trace the peak. It produces a number that is proportional to peak area.

Weigh

Triangulation
Main manual method. Assumes that each peak approximates a triangle. Area can be determined by area = peak height x width or area = peak height x 2 W1/2

Triangulation
Create an isosceles triangle and extrapolate the height and width. This is useful for regular shape peaks but where you might have peak overlap.

h w w

Triangulation

Integrating recorders
A special two pen recorder. The first pen tracks the chromatographic signal. The second traces a series of zigzags.

h1

h2

w1

w2

Integrating recorders
The larger the peak response gets, the more rapidly the second pen sweeps back and forth. The total number of zigs and zags can then be related to the peak area. If the peak gets to large, the second pen stops moving. You must keep the peak with in range.

Digital integrators
Relies on A/D conversion of detector response.

Peak recognition
width of a single A/D reading

Peak recognition
The sampling rate must be high enough so that the number of points represents the signal being measured.

A peak is initially subjected to A/D conversion. This results in a series of discrete measurements at known time internals.

This example shows what can happen if the sampling rate is too low compared to variations in the signal.

Peak recognition
Start of peak. We can evaluate the change in our data (first and second derivative) as a way of detecting the start of a peak. 1st and 2nd derivative are still positive OK - its a peak. 1st and 2nd derivative are zero no peak. 1st and 2nd derivative are positive possible peak.

Peak recognition
Top of peak. We need to know the point of RMAX.

positive slope

negative slope

We can look for a change in slope as a way of detecting the top of a peak. The true apex can be calculated by using a quadratic fit of the surrounding points.

Peak recognition
End of peak. Essentially the reverse of detecting the start of a peak. Typically, a system will look for a minimum slope for termination of a peak. The maximum peak width can also be used as a factor for ending a peak.

Peak area is then found by summing all of the readings. The baseline is corrected by subtracting the average of the first and last data points.

You dont always have baseline resolved peaks. Here, which method would be best - the black or green?

There are many options and parameters that you have available with modern integrators and integrating software.

Some common parameters

Peak width Too narrow - noise spike. Too wide - baseline shift. Threshold How far above baseline a signal needs to be to start a peak. Solvent peak Use tangent skimming to set baseline. With so many options and peak types, finding the right set is an art form.

Digital integrators
Caution! Peaks are typically process on the fly. If a peak is missed, the run must be repeated. This can happen even with the best methods.

Computer systems
Include the same methods of peak detection and integration as integrators. Major advantage is that the entire chromatographic run is stored prior to analysis. This allows you to test out various methods of integration on a single run and to reanalyze data if a peak is missed.
(or when your research advisor tells you what you did wrong!)

Summary

Method

Time, min 15 10 20 5 N/A N/A

Precision, % 4.1 2.5 - 4 1.7 1.3 0.44 0.44

Planimeter Triangulation Cut & weigh Int. Recorder Integrator Computer

Quantitative interpretation
OK, now you have all of your peak areas. Lets assume you knew what you were doing and all the areas were measured properly.

Determining concentration
Several approaches can be used. Use the one that is most appropriate for your method.

Big deal!
A relationship between concentration and area must be established or were just spinning our wheels.

Methods well cover Internal normalization External standard method Internal standard method

Internal normalization
Calculate the total area of all peaks in a sample and assume: Each component produces a peak Detector response is not concentration dependent The solvent peak, if any, is typically ignored.

Internal normalization
With these assumptions:

%Ci - % Area =

Areai Area total

This method is commonly reported as the default for integrators. Since most detectors give responses that are both concentration and substance dependent, the method only serves to give a ballpark estimate of relative concentrations.

External standard method

Requirements for proper use:

External standard method

You either assume that response is linear over the entire concentration range or actually measure it. Then:

! Standard solution containing all eluents to be quantified. ! Standard eluents should be of similar concentration as unknowns. ! The standard and sample matrix should be as similar as possible ! Analysis conditions must be identical stable instrument, same sample size ...

area concunk = area unk concunk known

This is assuming that the same injection volume was used for both the unknown and standard.

External standard method

Example - determination of X in MeCl2 Prepare a standard of X (20.0 mg in 100 ml MeCl2) - 0.200 g/l Use an injection volume of 5 l for both the standard and the unknown. Measure the areas produced by both the sample and the unknown. Area Xstd Area Xunk = 2000 units = 3830 units

External standard method

Now, determine the concentration of X in you unknown.

area concunk = area unk concunk known concunk = 3830 ng 0.200 nl 2000
ng

concunk = 0.384 nl

You can now convert to a more appropriate concentration if required.

Internal standard method

Overall, the most reliable approach. Basis A known substance is added at a constant concentration to all standards and samples - internal standard. Since the internal standard is always present at a constant amount, it can be used to account for variations such as injection volume during an analysis.

Internal standard method

Requirements for an internal standard. " Must be present at a constant concentration in all samples and standards. " Must be stable and measurable under the analysis conditions. " Must not interfere with the analysis or co-elute with sample components.

Internal standard method

Three common approaches are used Classical method - weighed portions of the standard and sample are combined Stock solution - a known volume of the sample is spiked with a known volume of the standard Calibration plot - a series of standards are run and a curve plotted based on corrected peak areas.

Internal standard method

Regardless of the method for introducing the standard or calibrating, the calculations are the same. Our NORM or ISTD substance is now predetermined and has a fixed value.

Internal standard method

Internal standard method

It is assumed that variations in the internal standard area are representative of the whole analysis from the point where it is introduced. The earlier, the better. Accounts for factors such as: Sample injection errors or changes Slow detector variations Slow column changes Variations in sample prep

Cunk =
Cunk Cknown AISTDknown AISTDunk Aunk Aknown

AISTD AISTD

known unk

Aunk C Aknown known

Amount of unknown Amount of known Area of internal standard in known Area of internal standard in unknown Area of unknown peak Area of known peak

Examples
Standard Too little injected

Internal standard method

Example Prepare a standard that contains 11.3 mg of X and 12.00 mg of ISTD. Make several 2 l injections and calculate an average response for each component.

Too much injected Unknown

Component X ISTD

Average area 635 1009

Internal standard method

Now, inject your unknown. AreaX = 990 1031

Internal standard plot method

" Hold the ISTD constant but vary the amount of the target species in a series of standards. " Create a calibration curve using the corrected areas. " Useful when the linearity of the detector is in question.

AreaISTD =

CX = (1009/1031) (990/635) x 11.3 mg = 17.24 mg X in the unknown.