LC Technology flashcards

40 cards. Read the term and the definition here. The deck at the top of this page is optional.

Retention factor k

k = (tR − t0)/t0, where tR is the analyte retention time and t0 the unretained marker time. Practical values are 1–10.

Resolution equation

Rs = 2(tR2 − tR1)/(w1 + w2). Baseline resolution is generally Rs ≥ 1.5; Rs of 1.0 gives about 2% overlap.

Van Deemter equation

H = A + B/u + C·u describes plate height versus linear velocity. The A term is eddy diffusion, B longitudinal diffusion, C mass transfer resistance.

Plate number N

N = 16(tR/w)² or 5.54(tR/w1/2)², measuring column efficiency. Higher N narrows peaks and improves resolution by the square root.

Selectivity factor α

α = k2/k1, the ratio of retention factors of two adjacent peaks. Selectivity is the most efficient lever for improving resolution.

Reversed-phase retention

Retention increases with analyte hydrophobicity and decreases as organic content rises. Ionizable analytes shift retention with mobile-phase pH.

HILIC retention mechanism

HILIC retains polar analytes on a polar stationary phase with high organic mobile phase. It requires at least 80% organic at start and aqueous-rich injection solvent.

Gradient LSS model

log k = log kw − S·φ relates retention to organic fraction φ. Gradient peak capacity depends on gradient time, flow, and column dead volume.

Degassing purpose

Removing dissolved gas from mobile phase prevents bubbles that cause pump noise, baseline spikes, and retention-time drift. Methods include helium sparge and in-line vacuum.

Autosampler carryover

Residual analyte from a prior injection contaminates the next run. Needle wash with strong then weak solvent and blank injections quantify and reduce it.

PDA peak purity

Photodiode array detectors compare spectra across a peak. Spectral match and absorbance ratio flag co-eluting impurities that share retention time.

ESI versus APCI

ESI ionizes polar and ionic analytes from solution; APCI suits nonpolar, low-polarity compounds. Mobile-phase pH and composition strongly affect ESI response.

Refractive index detector limits

The RI detector is universal but incompatible with gradient elution and sensitive to temperature. It offers lower sensitivity than UV or fluorescence detection.

Column oven stability

Temperature control to ±0.1–0.5 °C stabilizes retention and selectivity. Drift in oven temperature produces baseline drift and shifting retention times.

Extra-column volume

Tubing and fittings after the column add dead volume that broadens bands. Low-dead-volume connections preserve resolution, especially on narrow-bore columns.

Mobile-phase buffer choice

Phosphate buffers cover pH 2–8 but are nonvolatile. Ammonium formate and acetate are volatile and preferred for MS-compatible methods.

Method transfer scaling

Keep linear velocity constant when changing column diameter by scaling flow with d². Keep gradient volume constant when changing length or flow.

Forced degradation study

Acid, base, oxidation, heat, and light stress demonstrate specificity. Mass balance and peak purity confirm degradants are separated from the main peak.

System suitability criteria

Typical acceptance limits: resolution ≥ 1.5–2.0, tailing factor ≤ 2.0, replicate injection RSD ≤ 2.0%, and plates above a method-defined minimum.

ICH Q2 validation parameters

Accuracy, precision, specificity, linearity, range, detection and quantitation limits, and robustness, each with predefined acceptance criteria and statistical basis.

Injection volume rule

Injection volume should be no more than 1–2% of column volume. A weaker injection solvent than the mobile phase prevents peak distortion.

Protein precipitation

Adding at least three volumes of acetonitrile or methanol to sample precipitates protein. Centrifugation and filtration follow before injection.

SPE sequence

Condition, equilibrate, load, wash, and elute. Sorbent chemistry must match analyte polarity, and controlled flow prevents channeling.

Internal standard choice

A stable-isotope-labeled analog is preferred for LC-MS because it co-elutes and corrects recovery and ionization. A structural analog is the fallback.

Carryover acceptance

A blank injected after a high-concentration sample should show carryover below the method LOQ, commonly ≤20% of LOQ response.

Sample stability testing

Bench-top, refrigerated, freeze-thaw, and autosampler residence conditions are evaluated. Autosampler temperature affects volatile or degradable analytes.

LOD and LOQ formulas

LOD = 3.3σ/S and LOQ = 10σ/S, where σ is the standard deviation of the response and S the slope. Signal-to-noise of 3:1 and 10:1 also apply.

Calibration model selection

External standards suit clean matrices. Internal standards correct variable recovery and injection. Standard addition suits matrix-matched quantitation.

Linearity assessment

Use at least five or six calibration levels. Correlation coefficient alone is insufficient; residual plots reveal curvature and heteroscedasticity.

Data integrity ALCOA+

Records must be attributable, legible, contemporaneous, original, and accurate. Audit trails, unique accounts, and controlled electronic records support this.

High backpressure diagnosis

Disconnect the column and measure system pressure to locate the restriction. Clogged frits, guard columns, and inline filters are common causes.

Ghost peak causes

Carryover, contaminated mobile phase, or prior injections produce peaks absent from the blank. A blank gradient and fresh solvent isolate the source.

Peak tailing causes

Silanol interactions, a partially blocked frit, or column voiding produce tailing. Check pH, guard column condition, and fitting integrity.

Baseline drift causes

Inadequate degassing, lamp aging, incomplete equilibration, and temperature instability cause drift. A UV baseline drift often indicates a failing lamp.

Pump seal maintenance

Replace pump seals at manufacturer-defined intervals and verify check-valve function. Noisy pump traces often indicate a failing check valve or air.

Peroxide-forming solvents

THF, diethyl ether, dioxane, and isopropanol form peroxides over time. Date containers on receipt and test or discard before expiration.

HPLC waste classification

Solvent-rich HPLC waste is hazardous when ignitable or toxic. Segregate aqueous and organic streams and label with accumulation start date.

Fume hood face velocity

Hoods are typically maintained at 0.3–0.5 m/s (60–100 ft/min). Verify with an anemometer and confirm sash height markers before use.

Compressed gas cylinder safety

Cylinders must be secured at one-third and two-thirds height. Regulators are gas-specific; hydrogen service requires compatible equipment.

Chemical hygiene plan

The plan documents SOPs, engineering controls, PPE, training records, SDS access, and waste procedures for each laboratory method.