Introduction to Separations

Question 1

Peak Capacity

Answer 1

• the maximum number of peaks that can be theoretically separated on a column under given chromatographic conditions with R_S = 1
• can be calculated from the peak width (w) at 13.4% of the peak height and the separation (gradient) time (tg)
• P = 1 + (tg/w)

Question 2

Number of Theoretical Plates

Answer 2

• a measure of the analyte dispersion on the separation column

Question 3

Peak Asymmetry

Answer 3

• in the real world peaks would be gaussian with even distribution, however this is not always the case

Question 4

Peak Resolution (Two Methods)

Answer 4

• the separation of two peaks based on their retention times as well as their corresponding peak width at base (calculated by the triangulation method)
  
  • baseline peak width is 4σ
  • FWHM is 2.355σ
• R_S >=1.5 equates to baseline separation

Question 5

How Should We Separate? (Three Considerations)

Answer 5

1. What is the major difference in the analytes?
2. What kind of separation technique?
   
   • Liquid Chromatography - separation of molecules based upon differences in hydrophobicity, charge and size
   • Electrophoresis - separation of molecules based on differences in size-to-charge ratio, isoelectric point, hydrophobicity and molecular weight
3. What kind of detection?
   
   • dectection based upon analytes and their quantities

Question 6

Evaluating Separation Performance (Three Factors)

Answer 6

1. Resolution
2. Peak Capacity
3. Number of Theoretical Plates

Question 7

Peak Efficiency

Answer 7

• a measure of the dispersion of the analyte band as in travels through the column