Chromatography Notes

Flashcards covering key concepts and definitions in chromatography.

Chromatography

A separation technique where components partition between a mobile phase (moves) and stationary phase (fixed).

Mobile Phase

The phase that moves through the system (gas, liquid, or supercritical fluid).

Stationary Phase

The phase that stays fixed (solid support or liquid coating on solid).

tm

Dead time (void time) - the time for an unretained compound to pass through the column.

tr

Retention time - the time from injection to peak maximum.

Partition Coefficient (K)

K = Cs/Cm where Cs is concentration in stationary phase and Cm is concentration in mobile phase. Large K means longer retention.

Retention Factor (k)

k = (tr - tm)/tm. Measures how much a compound is retained relative to an unretained compound.

Optimal k Range

The optimal range for k is 2 < k < 10. Values below 2 give poor separation, values above 10 give long analysis times.

Selectivity Factor (α)

α = k2/k1 (where k2 > k1). Measures how different two compounds' retention is.

Improving Selectivity (α)

Change stationary phase, change mobile phase composition, or adjust temperature.

Number of Theoretical Plates (N)

N = 16(tr/wb)² = 5.545(tr/w1/2)². Measures column efficiency - higher N means narrower peaks and better separation.

Plate Height (H)

H = L/N where L is column length. Lower H means better efficiency.

Resolution (Rs)

Rs = 2(tr2-tr1)/(wb1+wb2). Measures separation quality.

Master Resolution Equation

Rs = (√N/4) × [(α-1)/α] × [k2/(1+k2)]. Shows resolution depends on efficiency (N), selectivity (α), and retention (k).

Van Deemter Equation

H = A + B/u + Cu where u is flow rate. Describes band broadening from three sources.

A Term in Van Deemter

Eddy diffusion - multiple paths through packing. Independent of flow rate.

B/u Term in Van Deemter

Longitudinal diffusion - diffusion along column axis. Dominates at low flow rates.

Cu Term in Van Deemter

Mass transfer - slow equilibration between phases. Dominates at high flow rates.

Optimal Flow Rate

The B/u and Cu terms balance, giving minimum H (best efficiency).

Advantages of Open Tubular (OT) Columns

A ≈ 0 (no packing), much higher N (100,000-500,000), lower H, lower pressure. Used in GC.

Advantages of Packed Columns

Higher sample capacity, shorter analysis time. Used in HPLC.

Effect of Smaller Particles on Efficiency

Smaller particles → lower A and C terms → lower H → higher N → better resolution.

Column Diameter and Retention

Larger diameter → lower k (more mobile phase volume) but higher H (longer diffusion distances). Generally lower resolution.

Effect of Doubling Column Length

N doubles, but Rs only increases by √2 = 1.41×. Analysis time doubles. Diminishing returns.

Quantitation Methods

External calibration (separate standards), standard addition (spike sample), internal standard (add same amount of IS to all).

Using External Calibration

Simple, fast, multiple analytes. Good for clean samples with accurate injection.

Using Standard Addition

For complex matrices (blood, soil, food). Corrects for matrix effects.

Using Internal Standard

For procedural variations (injection volume, detector drift). Add same amount of IS to all samples.

Good Internal Standard Characteristics

Chemically similar to analyte, well resolved from analyte, not present in samples, stable.

Fundamental Quantitation Principle

Peak Area ∝ Concentration (at detector). Larger peak area means higher concentration.

Optimizing Retention (k)

If k < 2: decrease temperature (GC) or solvent strength (HPLC). If k > 10: increase temperature or solvent strength.

Optimizing Selectivity (α)

Change stationary phase or mobile phase composition. This has the biggest impact on resolution.

Optimizing Efficiency (N)

Use longer column, optimize flow rate, use smaller particles.

GC Use

Gas Chromatography - for volatile compounds. Mobile phase is inert gas (He, N2).

HPLC Use

High Performance Liquid Chromatography - for non-volatile or thermally unstable compounds. Mobile phase is liquid solvent.

Fraction in Mobile Phase

Fraction in mobile = tm/tr = 1/(1+k).

Fraction in Stationary Phase

Fraction in stationary = (tr-tm)/tr = k/(1+k).

Rs = 1.0 Meaning

Peaks are touching, approximately 98% separated. Not baseline resolution.

Rs = 1.5 Meaning

Complete baseline resolution - this is the target for good separation.

Rs = 2.0 Meaning

Excellent separation with space between peaks.

Temperature Effect on GC Retention

Higher temperature → lower K → lower k → faster elution.

Solvent Strength Effect on HPLC Retention

Stronger solvent → lower k → faster elution. Weaker solvent → higher k → slower elution.

wb in Chromatography

Peak width at baseline, measured by tangent method. Used to calculate N.

w1/2 in Chromatography

Peak width at half height (50% of peak maximum). Also used to calculate N.

N = 16(tr/wb)² Calculation

Calculates the number of theoretical plates using baseline width method.

N = 5.545(tr/w1/2)² Calculation

Calculates the number of theoretical plates using half-height width method.

Peak Tailing Indication

Poor column condition, wrong pH, or need for column deactivation. Reduces efficiency.

Fixing Poor Retention (k < 2)

Decrease temperature (GC) or use weaker solvent (HPLC) to increase k to optimal range 2-10.

Fixing Excessive Retention (k > 10)

Increase temperature (GC) or use stronger solvent (HPLC) to decrease k to optimal range 2-10.

Coeluting Peaks Problem (α = 1)

Change stationary or mobile phase to get α > 1.1 for separation.

Causes of Broad Peaks (Low N)

High H from: wrong flow rate, dead volume, poor packing, or column degradation.