1. Lecture 8 Peak Parameters and Quantitative chromatography
PHCMt561 Instrumental Analysis - WS 10/11 - Lecture 8
Lecture 8 Peak Parameters and Quantitative chromatography
Dr. Rasha Hanafi
© Dr. Rasha Hanafi, GUC
Lecture 8 – Chromatography,
Dr. Rasha Hanafi,

2. Lecture 8 – Chromatography, 30-10-2012
Objectives
To define peak parameters:
retention time, dead time, peak width, tailing factor, retention factor, separation factor, plate number and resolution.
To compare isocratic elution and gradient elution.
To quantify compounds using their chromatograms.
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Lecture 8 – Chromatography,

3. Lecture 8 – Chromatography, 30-10-2012
Reminder
When a sample is injected, 3 things occur:
The components of the sample are carried out by the mobile phase through the column, i.e. SAMPLE MIGRATION. The rate of migration of any particular sample component is determined by that component’s retention by the system.
Different components of the sample migrate at different rates through the column. The DIFFERENTIAL MIGRATION forms the basis of a chromatographic separation.
As bands of sample components migrate through the column, they broaden. This BAND BROADENING limits the resolution that can be obtained between adjacent bands.
A BAND is the actual physical distribution of sample molecules within the column. A PEAK is the graphic representation of that band on a recorder or data system as it elutes from the column.
© Dr. Rasha Hanafi, GUC
Lecture 8 – Chromatography,

4. PHCMt561 Instrumental Analysis - Lecture #7
1.1. Retention of a peak
time
I (detector signal intensity)
tR1 tM
tR'= tR1- tM
tR2
tR2'= tR2- tM
Retention time tR is the time from injection to the appearance of the highest point of the peak. When all conditions are held constant, tR for a compound remains constant, and serves for qualitative analysis by comparing it with a reference pure substance (c.f. Rf in TLC).
Retention volume VR is the volume of the mobile phase that must be passed through the system in order to elute the peak. “VR = tR x flow rate”
Dead time tM is the time it takes for an unretained component to pass through the column void volume= tM x flow rate. tM is easily estimated from the small disturbance that marks the 1st feature of the chromatogram.
© Dr. Rasha Hanafi, GUC
Lecture 8 – Chromatography,
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5. 1.1. Retention of a peak, cont.
Retention factor (K’) is the no. of column volumes of mobile phase required to elute a band after the initial volume of the column has been displaced. K’= (tR-tM)/tM.
K’ doesn’t change when flow rate or column length are changed (check the formula to know the reason?).
Separation factor (α): it shows the ability of a certain chromatographic system to discriminate between 2 components (2 peaks).
α = K’2/K’1= t’R2/t’R1
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Lecture 8 – Chromatography,

6. 1.2. Width and Height of a peak
PHCMt561 Instrumental Analysis - Lecture #7
1.2. Width and Height of a peak
Width at baseline wb.
Width at half height w0.5 (more accurate measurement).
For one and the same concentration of a substance, do you think that peak height and width change, if they do, what could be considered constant?
© Dr. Rasha Hanafi, GUC
Lecture 8 – Chromatography,
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7. 1.3. Parameters of peak asymmetry
Tailing factor Tf is a measure of peak shape. A well-behaved peak appears as bell-shaped or Gaussian. Yet, peaks may show FRONTING or TAILING. What could be the reason for either?
Asymmetry factor Af is defined as “BC/CA “at 10% of the peak height.
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Lecture 8 – Chromatography,

8. 1.4. Assessment of separation, Plate number
PHCMt561 Instrumental Analysis - Lecture #7
1.4. Assessment of separation, Plate number
As the sample bands move through the column, they broaden,
Why?
The more plates, the better the separation! The lower the plate height, the better the separation!, How to decrease plate height?
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Lecture 8 – Chromatography,
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9. 1.5. Assessment of separation, Resolution
PHCMt561 Instrumental Analysis - Lecture #7
1.5. Assessment of separation, Resolution
Resolution (R) defines the degree of separation of 2 adjacent bands. Base line resolution is achieved when R>1.5 and it is a fundamental goal for quantitative analysis.
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Lecture 8 – Chromatography,
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10. Performed with a constant solvent mixture.
2.Types of elution
Isocratic
Performed with a constant solvent mixture.
Gradient
Increasing amounts of one of the components are added to create a continuous gradient
Linear
Stepwise
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Lecture 8 – Chromatography,

11. 2.1. Drawbacks of Isocratic Elution
PHCMt561 Instrumental Analysis - Lecture #7
2.1. Drawbacks of Isocratic Elution
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Lecture 8 – Chromatography,
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12. PHCMt561 Instrumental Analysis - Lecture #7
2.2. Stepwise Gradients
How to combine all the advantages of the different mobile phases (fast elution and good signal shape – in the previous example: high %B and good separation – in the previous example: low %B) in one chromatographic system?
“Stepwise" or “Segmented" gradient
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Lecture 8 – Chromatography,
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13. PHCMt561 Instrumental Analysis - Lecture #7
2.3. Linear Gradients tD
Linear gradient
“Dwell time" tD: time required for the gradient to reach the column, from "dwell volume" VD: volume between the point where solvents are mixed and the beginning of the column.
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Lecture 8 – Chromatography,
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14. 3.1. Peak area, Quantitative analysis
Detector signal is directly proportional to analyte concentration. If we want to know the total amount of an analyte, we should relate a series of concentrations to their respective Area under the curve (AUC) via a calibration curve. Concentration α AUC
Determination of the AUC: calculated by the software (baseline must be defined) or manually: if shape is Gaussian: AUC = x h x w0.5
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Lecture 8 – Chromatography,

15. Lecture 8 – Chromatography, 30-10-2012
3.2. Internal Standards
It is a substance added with the same amount of same compound to all calibration and samples. Then the ratio of area of compound to area of the internal standard is used as the Y-axis in the calibration curve. Internal standards are especially useful for analyses in which the quantity of sample analyzed or the instrument response varies slightly from run to run for reasons that are difficult to control. A calibration curve is only accurate for the one set of conditions under which is obtained. If the relative response of the detector to the analyte and standard increases, the signal from the analyte also increases by the same extent . As long as the concentration of standard is known, the correct concentration of analyte can be derived. Internal standards are widely used in HPLC because the small quantity of sample solution injected into the HPLC is not very reproducible in some experiments. Internal standards are also desirable when samples loss can occur during sample preparation steps prior to analysis. If a known quantity of standard is added to the unknown prior to any manipulation, the ratio of standard to analyte remains constant because the same fraction of each is lost in any operation. Properties of an internal standard: chemically inert, similar chemical structure from the analyte, must have resolution and detectability and mimics analytes in pretreatment steps.
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Lecture 8 – Chromatography,

16. Lecture 8 – Chromatography, 30-10-2012
References
“Principles of instrumental analysis, 5th ed. by Skoog, Holler, Nieman” Chapter 26.
“Quantitative Chemical Analysis, 7th ed. By Harris” Chapter 25.
Lecture of “Chromatography-II” by Dr. Raimund Niess, GUC, 2009.
Reference Guide of the DryLab® software, Molnár Institute for Applied Chromatography, Berlin.
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Lecture 8 – Chromatography,

17. Lecture 8 – Chromatography, 30-10-2012
Now it is time for Quiz 2!! Good luck Reminder of Locations 9:15 to 9:45 AM
Location
Group
Lecture Hall H5
T 2
T 6
T 7
Biotechnology group
Lecture Hall H1
T 1
T 8
Exercise Room - C5.105
T 9
Exercise Room - C5.204
T 3
Exercise Room - C5.206
T 4
Exercise Room - C5.209
T 5
Exercise Room - C5.210
T 10
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Lecture 8 – Chromatography,