Chromatography Basics

Chroma = Color Graphy = to write

Dr. Suneel Kumar Onteru Animal Biochemistry Division National Dairy Research Institute Karnal, India

What is chromatography?

Chromatography is a separation technique that uses the size, shape, chemical properties or charge of molecules in a sample to separate the sample into its constituent components. It is often used to detect one, or a number of, components in a complex mixture
2

What is chromatogram?
The visual graph or picture obtained from a chromatography experiment

Absorbance

Retention time
3

Basic requirements of chromatography

Mobile Phase

Reservoir for mobile phase

Stationary Phase

Collector
4

Basic requirements of chromatography

Sheehan, D. 2009. Physical Biochemitsry-Principles and applications, 2nd edition, Wiley 5 and Blackwell publishers, West Sussex, UK. Page No. 13.

Properties of stationary phase

Made up of solid particles or solid particles coated with liquid Solid particles: Offer good flow characteristics Possess sufficient mechanical strength Chemical inertness for biomolecules to be separated Chemical groups are covalently attached to solid particles in bonded-phase chromatography e.g., cellulose or agarose Solid particles coated with liquid Liquid is attached by non-covalent attraction (liquidliquid chromatography) E.g., silica coated with non-polar hydrocarbon 6

Properties of Mobile phase

Aqueous buffers for retaining native structure of biomolecules
Reason: Biomolecules are evolved to function in aqueous environment

Organic solvents if native structure of biomolecules is not required Inert gases for volatile compounds

Basic principle of Chromatography

Partition coefficient
KD = Solute concentration in mobile phase / Solute concentration in stationary phase KD depends on Physico-chemical interactions among sample, mobile and stationary phases Experimental conditions e.g., temperature, solvent polarity Efficient separation of biomolecules depends on the exploitation of tiny differences in their partition coefficients
8

Common physico-chemical interactions (Or) Modes (Or) Retention mechanisms in chromatography

Solubility - Partition chromatography Adsorption - Adsorption chromatography Size - Size exclusion chromatography Attraction between opposite charge - Ion-exchange chromatography Biospecific interaction with ligand - Affinity chromatography
9

Performance parameters in chromatography

Useful to compare different columns when replaced one with another 1. Retention of solutes: Measured by retention volume (VR) in ml retention time (tR) in minutes VR = f. tR f = flow rate of mobile phase (ml/min)

10

Performance parameters in chromatography

1. Retention of solutes: Example
(Retention volume for solute 2) (Retention volume for solute 1)

(Void volume)
Peak width

Peak area indicates quantity of solute

Retention time

Sheehan, D. 2009. Physical Biochemitsry-Principles and applications, 2nd edition, Wiley 11 and Blackwell publishers, West Sussex, UK. Page No. 14.

Performance parameters in chromatography

Void volume (Vo): Volume required for the solvent or mobile phase to pass, unretained through the column and eluted Volume of column plus injection valve and tubing minus volume of packaging material in column 2. Retardation factor (RF) of solute RF = Rate of movement of solute / Rate of movement of mobile phase Useful more in planar chromatography like paper and thin layer chromatography
12

Performance parameters in chromatography

3. Resolution (R): How two peaks are resolved from each other Useful to compare how effective a given column is in separating a mixture
V2 - V1 R= (W1 + W 2) / 2
Narrow (i.e small W) and well separated (e.g., large V2V1) peaks indicate best chromatography
13

Performance parameters in chromatography 4. Peak broadening:

Elution of sample in a much larger volume than that in which it was applied due to a phenomenon called band broadening Effects resolution (R) Band broadening could be due to
Diffusion of the sample due to Eddy diffusion Mass transfer phenomenon Mobile phase mass transfer Stagnant mobile phase mass transfer Stationary phase mass transfer
14

Performance parameters in chromatography

4. Peak broadening:
Eddy diffusion Mobile Phase mass transfer

Variation in size of stationary phase particles causes narrow and Broad channels Whirlpools or Eddies of mobile phase in narrow channels cause wide distribution of sample between fast flowing wide and slow flowing narrow channels

15

Performance parameters in chromatography

4. Peak broadening:
Stagnant mobile phase mass transfer Stationary phase mass transfer

Channels not oriented in same direction results in stagnation of mobile phase in some parts of the column

16

Performance parameters in chromatography

5. Theoretical plates in column: The number of equilibrations that a compound makes with the stationary phase Columns are considered to consist of a number of adjacent plates or zones where there is enough space for a compound to achieve complete equilibrium between the mobile and stationary phase. Each zone is called a theoretical plate and the length of the column the plate height.
17

Performance parameters in chromatography

5. Theoretical plates in column:

18

Performance parameters in chromatography

5. Theoretical plates in column:

19

Performance parameters in chromatography

5. Theoretical plates in column:

20

Performance parameters in chromatography

5. Theoretical plates in column: Effect of Kd on chromatographic separation

21

with the stationary phase. 5.Chromatography Theoretical plates columns in are considered to consist of a number of column: adjacent plates or zones where there enough space for a compound to is The more theoretical plates achieve complete equilibrium between the mobile stationary of the better the and resolution phase. Each zone is called a solute plate and the length of the theoretical column the plate height. The more t Rthe2better the theoretical plates N = 16.( ) Consider three resolution of protein. W a different number columns each with of theoretical plates

chromatography Performance parameters in Theoretical Plates the number of chromatography equilibrations that a compound makes

W
Relative distribution on the column

tR 2 N = 5.54.( ) Wh

N = No. of theoretical plates

22

Wh = Width at half the height of a peak

Performance parameters in chromatography

5. Theoretical plates in column: N is directly related to surface area of the particles in stationary phase Plate height H = Length of the column / N Different length columns may have same number of theoretical plates Small H leads to better chromatography separation
23

Performance parameters in chromatography

5. Theoretical plates in column: Empirical relationship between plate height and flow rate of mobile phase (v)

B H = A + + C.v v
H = Plate height A = Eddys diffusion B = Longitudinal diffusion (Diffusion along with direction of flow) C = Mass transfer effects
24

Performance parameters in chromatography

5. Theoretical plates in column: Van Deemter curve Decrease in C.v gives smaller H, which
results in more N and better chromatography Extremely slow rates increases H due to increase in B/v (longitudinal flow), decrease in N and so poor chromatography A balance between C.v and B/v is the optimum rate (v) Low flow rate results long analyses times Higher flow rate than optimum leads shorter analyses times with inefficient separation 25

Performance parameters in chromatography

6. Capacity factor (k) and separation factor ()
Capacity factor for component 1 V1 = V0 (1+ k1)
Capacity factor for component 2 V2 = V0 (1+ k2) Separation factor = k1/k2 High separation factor denotes good chromatography

26

Performance parameters in chromatography

7. Peak symmetry Ideal condition is Guassian curve or normal curve without any overlaps Deviations are common in practical chromatography due to non-linear flow rates and gradient elution methods.

27

Significance of performance parameters in chromatography

As the performance parameters depends primarily on physical interactions between sample components and stationary phase, these cane be used to compare different chromatography systems (e.g., HPLC and ionexchange chromatography) as well as different sample components (e.g., peptides and sterol derivatives)
28

A good chromatography needs..

High resolution (R) A large number of theoretical plates (N) in a column A low plate height (H) High values for separation factor ()

29

Reference
Most of this material is prepared fromSheehan, D. 2009. Physical BiochemitsryPrinciples and applications, 2nd edition, Wiley and Blackwell publishers, West Sussex, UK. Page No. 11-50.

30