1. The Chromatogram  Terms:  Retention time  Peak area  Peak width (at half height, at base)  Peak height  Void time/volume  Adjusted retention time  Leading edge, tailing edge

2. The chromatogram TERMS - Retention time, Peak Area, Void Time (volume), Adjusted Retention Time Peak Width ( at half height, at baseline), Peak Height, Leading Edge, Tailing Edge

3. Chromatographic Terms  Retention volume  Volume of mobile phase needed to carry component through column to detector  V R

4. Retention volume  Not convenient to measure volume directly  Measure time taken from injection to appearance of signal peak  Record as retention time t R -maintain a constant flow rate V R = t R Fc where Fc = flow rate

5. Retention time

6. Void volume/time  A compound cannot possibly exit the column in “zero seconds”  Takes time to travel through a column even at “top speed”  Fastest a component can travel is at the speed of the eluting solvent – ie the same speed as the mobile phase

7. Void volume/time  Have a flow rate of for example 1 mL/min  Column and tubing occupy a volume of 2 mLs of liquid (excluding volume occupied by solid phase)  Then fastest a component could travel would be 2 minutes – as fast as the MP

8. Void Volume/time  The fastest speed an unretained component could travel through the column and “system” is represented by the void volume/time  No peak can be seen before the void time  If a column has a void time of 2 minutes, then no peak can occur at <2 minutes

9. Void volume/time  To measure void time, inject a component known not to stick or be retained on your column  The peak you see will represent the void  Why is this important? i.e.Why is it important to an analyst to know the void time of their system when performing an analysis?

10. The adjusted retention time  Retention time = the time taken for a component to travel through the column  However, this cannot be less than the void time  Therefore the adjusted retention time is the actual retention time minus the void time

11. Adjusted retention time  V’ R = V R – V o  t’ R = t R – t - equation for calculating adjusted RT  Retention times and volumes are dictated by the distribution coefficient K  K= Cs/Cm

12. The chromatogram TERMS - Retention time, Peak Area, Void Time (volume), Adjusted Retention Time Peak Width ( at half height, at baseline), Peak Height, Leading Edge, Tailing Edge

13. Capacity Factor  k – describes the ability of the stationary phase to retain components  Ratio between number of molecules in SP compared to MP  k=Cs/Cm

14. Capacity Factor  k 1 = (V 1 -V o )/V o  k 1 = (t R –t o )/t o = t’ R /t o The longer a component is retained by the column, the larger the capacity factor

15. Capacity factor  Large capacity factors favour good separation but increased elution times  Capacity factors > 1 and < 5 are favoured  If over time the capacity factor changes, usually indicates degradation of the column

16. Separation Factor Selectivity  A purpose of chromatography is to separate compounds from each other  If want to separate A from B, they must have different retention times (ie different capacity factors)

17. Retention time

18. Selectivity  Separation factor (alpha) aka selectivity - for two peaks:  Alpha (a) = k B /k A = t’ R(B) /t’ R(A) For separation to occur, alpha must be greater than 1 Which peak must have the longer retention time?

19. Selectivity  Separation factor depends on type and properties of stationary phase used, the composition and properties of mobile phase, the interactive forces of the analyte and the column temperature  Must be optimized for each separation  If maximize alpha, get good separation BUT analysis times are too long

20. Separating efficiency of a column  Every component should show up as a nice narrow peak  Must then move in a narrow band through the column  Peak width is an indication of the efficiency of a column

21. Efficiency  Observation: the longer a component stays on the column, the wider the peak  The band is dispersed as it travels through the column  The longer it is on the column, the more time it has to disperse  Dispersion leads to band broadening and wide peaks

22. Peak width  Simple measurement: peak width  Gives an estimation of the efficiency of the column when combined with retention time  The number of theoretical plates, or the plate count, N is used as a quantitative measure of efficiency

23. Plate theory (again)  Chromatography is a continuous process  Separation occurs on all particle surfaces  Think of in terms of imaginary segments of the column called plates – remember the molecular “hurdles”?  Each plate represents a surface at which the separation occurs (“separation event” – ie the analogy of the hurdles in a race)

24. Plates  The more plates (hurdles) the better the separation and the efficiency of the column  Abstract concept used to compare the efficiencies of different columns  To give it a value, need two easy measurements, retention time and peak width

25. Plate count N  Relationship between Rt and W  As the retention time increases, so does the peak width  If look at two columns, compare peak width of peaks having the same retention time  Can use W or W1/2

26. Formula for N – Theoretical Plates  N = 16(V R /W) 2 = 16(t R /W) 2 Where both t R and W are in the same units (eg seconds or minutes) Remember, N is a measure of the separation power (efficiency) of the chromatographic system

27. Measurements for N