Chromatographic Analytical Methods

Basic Principle of Chromatography

Chromatographic methods separate the components of a mixture because the components distribute differently between:

• a stationary phase (fixed in place), and
• a mobile phase (moves through or along the stationary phase).

A component that interacts more strongly with the stationary phase moves more slowly; a component that prefers the mobile phase moves faster. This difference in migration speed leads to separation in time and/or space, which can be used for qualitative and quantitative analysis.

Common analytical goals:

• Identify which substances are present (qualitative analysis).
• Determine how much of each substance is present (quantitative analysis).
• Purify substances (preparative chromatography – mentioned here only briefly).

Types of Chromatographic Methods (Overview)

Chromatographic methods differ mainly in:

• the physical state of the mobile phase (gas or liquid),
• how the sample is introduced and detected,
• the scale (analytical vs. preparative),
• typical applications.

The main types relevant in instrumental analysis are:

• Gas chromatography (GC) – gas mobile phase
• High-performance liquid chromatography (HPLC) – liquid mobile phase under high pressure
• Thin-layer chromatography (TLC) – liquid mobile phase, stationary phase on a plate, usually simple and inexpensive
• Ion chromatography, size-exclusion (gel permeation) chromatography, affinity chromatography, etc. – specialized variants of liquid chromatography.

In this chapter we focus on the principle, the most common techniques (GC, HPLC, TLC), and how analytical information is obtained from chromatograms.

Retention and the Chromatogram

When a mixture is separated chromatographically, the detector records a signal versus time (or versus distance on a plate). This record is called a chromatogram.

Typical characteristics:

• Each peak corresponds ideally to one component.
• The retention time $t_R$ of a peak is the time between sample injection and the maximum of the peak.
• The dead time $t_0$ (or void time) is the time a non-retained species (one that only travels with the mobile phase and does not interact with the stationary phase) needs to pass through the system.

From these, a retention factor (also called capacity factor) can be defined (for column chromatography):

$$
k = \frac{t_R - t_0}{t_0}
$$

• $k$ gives a measure of how strongly a substance is retained by the stationary phase relative to the mobile phase.
• Larger $k$ means stronger interaction with the stationary phase and longer retention.

On a chromatogram you will also encounter:

• Peak area – proportional to the amount (or concentration) of the component, within the linear range of the detector.
• Peak height – sometimes used for quantification, but more sensitive to peak shape and conditions.
• Baseline – the signal when no analyte is eluting; an ideal baseline is flat and noise-free.

Parameters Describing Separation Quality

To judge how well a chromatographic separation has worked, some characteristic quantities are used.

Resolution

The resolution $R_s$ describes how well two neighboring peaks are separated:

$$
R_s = \frac{2\,(t_{R,2} - t_{R,1})}{w_1 + w_2}
$$

with:

• $t_{R,1}$ and $t_{R,2}$ – retention times of peak 1 and 2,
• $w_1$ and $w_2$ – peak widths at the baseline or at a defined height.

Typical interpretation:

• $R_s < 1$ – peaks are strongly overlapping.
• $R_s \approx 1.5$ – peaks are almost completely baseline-separated (good analytical resolution).

Column Efficiency

In column chromatography, efficiency reflects how narrow the peaks are for a given column length. It is often expressed via the number of theoretical plates $N$:

$$
N = 16 \left( \frac{t_R}{w} \right)^2
$$

• Higher $N$ means a more efficient column (sharper peaks).
• $N$ depends on the column, the conditions (flow rate, temperature), and the analyte.

Often the height equivalent to a theoretical plate (HETP), $H$, is used:

$$
H = \frac{L}{N}
$$

• $L$ – column length.
• Smaller $H$ means better efficiency (more plates per unit length).

The dependence of $H$ on the mobile-phase velocity $u$ is frequently represented by a so-called van Deemter curve, which shows an optimum flow rate for best efficiency (minimum $H$). The shape and origin of the curve are typically discussed in more advanced courses; here it is sufficient to know that:

• too slow or too fast flow rates reduce efficiency,
• there is an intermediate, optimal flow rate for best separation.

Modes of Separation: Adsorption, Partition, Ion Exchange, and Size Exclusion

The way substances interact with the stationary phase defines the separation mechanism:

• Adsorption chromatography
  Components adhere more or less strongly to the surface of a solid (e.g., silica gel, alumina). Often used in TLC and some normal-phase HPLC.
• Partition chromatography
  Components distribute between a mobile phase and a liquid-like stationary phase (e.g., a thin liquid film on a solid support). Typical for many reversed-phase HPLC and gas–liquid chromatography.
• Ion-exchange chromatography
  The stationary phase carries charged groups. Ions from the sample exchange with counterions on the stationary phase depending on charge and affinity.
• Size-exclusion (gel permeation) chromatography
  Separation is based primarily on molecular size. Large molecules are excluded from pores and elute earlier; small molecules enter pores and elute later.

Analytical methods often select the mechanism that best distinguishes the molecules of interest (by polarity, charge, size, or specific interactions).

Gas Chromatography (GC)

Principle

In gas chromatography:

• The mobile phase is an inert carrier gas (e.g., helium, nitrogen, hydrogen).
• The stationary phase is a thin liquid or polymer film on the inside of a long, narrow column (gas–liquid chromatography) or a solid adsorbent (gas–solid chromatography).
• The separation occurs at elevated, controlled temperatures.

Only substances that are sufficiently volatile and thermally stable can be analyzed directly by GC.

Main Components of a GC Instrument

• Carrier gas supply – provides the mobile phase at controlled flow.
• Injector – introduces the sample into the gas stream; the sample is quickly vaporized.
• Column – typically a capillary column, several meters long, inside a temperature-controlled oven.
• Oven – allows isothermal operation or temperature programming (gradual increase in temperature during the run).
• Detector – converts the amount of eluting substance into an electrical signal.

Common GC detectors:

• Flame ionization detector (FID) – very sensitive for organic compounds; measures ions formed in a flame.
• Thermal conductivity detector (TCD) – universal but less sensitive; measures changes in thermal conductivity of the gas mixture.
• Electron capture detector (ECD) – highly sensitive for halogen-containing compounds and some other electronegative analytes.

Variables Influencing Separation

• Column temperature
  Higher temperatures decrease retention times (faster analysis) but can reduce separation if peaks become too close.
  Temperature programming helps separate both low- and high-boiling components in one run.
• Carrier gas flow rate
  Affects efficiency and analysis time. Optimal flow is chosen based on column properties and gas type.
• Stationary phase polarity
  “Like dissolves like”: polar stationary phases retain polar compounds more strongly; nonpolar phases prefer nonpolar analytes.

Typical Applications of GC

• Analysis of volatile organic solvents and fuels.
• Monitoring of environmental pollutants (e.g., some pesticides, volatile organic compounds in air).
• Determination of flavor and fragrance compounds in food and beverages.
• Quality control of gases and gas mixtures.

High-Performance Liquid Chromatography (HPLC)

Principle

In HPLC:

• The mobile phase is a liquid (aqueous, organic, or a mixture).
• The stationary phase is finely divided solid particles in a packed column (or monolithic structures).
• High pressures (up to hundreds of bar) are used to push the mobile phase through the column.

HPLC is suitable for substances that are:

• non-volatile or thermally unstable (cannot be analyzed by GC),
• moderately polar to highly polar (depending on the mode),
• of medium to relatively high molecular mass (although very large biomolecules may require specialized techniques).

Main Components of an HPLC System

• Pump – delivers mobile phase at a constant and adjustable flow under high pressure.
• Injector (or autosampler) – introduces a defined volume of sample into the mobile phase.
• Column – typically stainless steel, packed with particles of specific size and chemistry.
• Detector – records the eluting components.

Typical detectors:

• UV/Vis absorbance detector – the most common; measures how strongly a substance absorbs light at selected wavelengths.
• Diode-array detector (DAD) – records the entire UV/Vis spectrum, aiding identification.
• Fluorescence detector – highly sensitive for naturally fluorescent substances or those derivatized to fluoresce.
• Refractive index (RI) detector – universal but less sensitive; often used for substances without strong UV absorption.

Normal-Phase and Reversed-Phase HPLC

Two important modes of HPLC:

• Normal-phase HPLC
• Stationary phase: polar (e.g., bare silica).
• Mobile phase: less polar organic solvents (e.g., hexane with polar modifiers).
• More polar analytes are retained longer.
• Reversed-phase HPLC (by far the most widely used)
• Stationary phase: nonpolar or weakly polar (e.g., silica with bonded C18 chains).
• Mobile phase: more polar (water mixed with organic modifiers like methanol or acetonitrile).
• Less polar analytes are retained longer; increasing organic solvent content usually decreases retention.

Isocratic and Gradient Elution

• Isocratic elution – mobile-phase composition is constant during the run.
• Simpler and more stable, but may be slow or insufficient for complex mixtures.
• Gradient elution – mobile-phase composition is changed during the separation (e.g., increasing organic content in reversed-phase HPLC).
• Allows good separation of components with very different polarities in reasonable time.
• Changes retention behavior over time: early peaks may be more polar, late peaks more nonpolar.

Applications of HPLC

• Analysis of pharmaceuticals and their impurities.
• Determination of vitamins, preservatives, and other additives in food.
• Analysis of pollutants in water and soil extracts.
• Separation and quantification of biomolecules like small peptides, nucleotides, and some metabolites.

Thin-Layer Chromatography (TLC)

Principle

In TLC:

• The stationary phase is a thin layer (typically silica gel or alumina) spread on a plate (glass, aluminum, or plastic).
• The mobile phase is a solvent or solvent mixture that rises up the plate by capillary action.
• Samples are applied as small spots near the bottom edge; the plate is placed in a chamber with a shallow pool of mobile phase.
• As the solvent moves upward, components move with it at different speeds, forming separated spots.

TLC is less automated than GC or HPLC, but it is:

• simple, inexpensive, and fast,
• well suited for qualitative checks (e.g., purity, reaction monitoring),
• used as a preparative method at small scale in some labs.

Retention Factor in TLC

In TLC, the retention factor (often denoted $R_f$) is used:

$$
R_f = \frac{\text{distance traveled by the substance (center of the spot)}}{\text{distance traveled by the solvent front}}
$$

• $R_f$ values lie between 0 and 1.
• Under identical conditions, each substance has a characteristic $R_f$.
• Changing the stationary phase, solvent system, or temperature will change $R_f$ values.

Visualization

Many substances are not visible on the plate without treatment. Common ways to visualize spots include:

• UV light – fluorescent indicator in the plate allows dark spots to appear where analytes absorb UV.
• Chemical staining – spraying the plate with reagents that react with specific functional groups to yield a colored or fluorescent product (e.g., ninhydrin for amino acids).

Typical Uses of TLC

• Quick check of reaction progress in synthesis laboratories.
• Identification of common components by comparing $R_f$ and spot appearance with reference substances.
• Rough assessment of mixture complexity before more sophisticated chromatographic analysis.

Quantitative Analysis with Chromatography

Chromatographic methods are widely used for quantitative analysis, especially GC and HPLC.

Calibration

To quantify an unknown, a calibration curve is often constructed:

1. Prepare standard solutions of the analyte at known concentrations.
2. Analyze each standard under identical chromatographic conditions.
3. Plot the detector response (usually peak area) vs. concentration.
4. Fit a line (or curve) to obtain a calibration function.

Then:

• Analyze the unknown sample.
• Measure the peak area of the analyte.
• Use the calibration function to find the corresponding concentration.

In many instruments, software carries out these steps automatically.

Internal Standard Method

To improve accuracy and correct for variations in:

• injection volume,
• detector sensitivity,
• sample preparation losses,

an internal standard can be used:

• A substance not present in the sample is added in a known amount.
• The ratio of analyte peak area to internal standard peak area is used for calibration and quantification.

This is common in GC and HPLC when high precision is required.

Detection Limits

Key quantitative parameters:

• Limit of detection (LOD) – the lowest amount or concentration that produces a signal distinguishable from noise (often defined as a signal-to-noise ratio of 3:1).
• Limit of quantification (LOQ) – the lowest amount or concentration that can be quantified with acceptable precision (often defined as a signal-to-noise ratio of 10:1).

Instrument sensitivity, background noise, and separation quality all influence these limits.

Chromatographic Selectivity and Method Optimization

For analytical work, it is often necessary to optimize a chromatographic method to:

• improve resolution between critical peak pairs,
• shorten analysis time,
• adjust to new analytes or sample matrices.

Important adjustable factors include:

• Stationary phase – its polarity, surface chemistry, and particle size.
• Mobile phase – composition, pH, ionic strength (in liquid chromatography).
• Temperature – particularly in GC, but also in some HPLC methods.
• Flow rate – influences efficiency and analysis time.
• Column length and diameter – affect resolution, run time, and pressure (in LC) or required flow rate (in GC).

Selectivity refers to how differently the method treats various analytes. Changing the stationary or mobile phase chemistry (for example, switching from reversed-phase to ion-exchange conditions) often has the greatest effect on selectivity.

Advantages and Limitations of Chromatographic Methods

Advantages

• High sensitivity and selectivity, especially with modern detectors.
• Capability to separate complex mixtures into individual components.
• Versatility – applicable to gases, liquids, and many types of solids (after dissolution or extraction).
• Combination with other instruments (e.g., GC–MS or LC–MS) for powerful qualitative and quantitative analysis.

Limitations

• Require careful method development and optimization.
• Often involve sample preparation (dissolution, filtration, extraction, derivatization).
• Columns and stationary phases have limited lifetimes and can be damaged by inappropriate samples.
• Not always suitable for extremely high-molecular-weight or highly reactive substances without specialized techniques.

Chromatographic analytical methods are therefore central tools in modern chemistry, enabling both routine quality control and advanced research across environmental, pharmaceutical, biological, and industrial applications.