GC & LC

Chromatography

What is the analytical technique that separates the components of a mixture so they can be examined individually?

Mobile, stationary

What are the two phases of chromatography?

1. ______: Moves through the system and carriers sample components forward. Is a gas in GC and a liquid in LC.

2. ______: Remains in place inside the column and interacts with the sample components as they pass through.

greater separation

More time in the mobile phase and a stronger interaction with the stationary phase will lead to what?

X=time, Y=detector response

What do the X and Y axis of a chromatogram represent?

Retention time

What is the time elapsed from a sample injection to the top of a chromatographic peak?

Peak height, peak area

Label the chromatographic measurements…

1. ______: The detector response as the highest point of the peak. Can change substantially if a peak becomes broader or narrower

2. ______: The total detector response across the entire peak. Commonly used for quantitative analysis because it accounts for the full signal rather than only its entire point.

Surface area/total area X 100

How do you calculate % composition?

A = 40%, B = 10%, C = 50%

Calculate percent composition…

• Area A retention of 0.8 minutes = 40

• Area B retention of 4.2 minutes = 10

• Area C retention of 0.8 minutes = 50

Resolution

What describes how well two nearby peaks are separated on a chromatogram?

Co-elution

What happens when two or more compounds exit at nearly the same time, and depending on the detector, they may appear as the same peak.

GC

What chromatography technique does this describe?

• A small amount of sample enters a heated inlet, where suitable compounds vaporize

• An inert carrier gas moves the vaporized compounds through the column

• Compounds separate because they differ in volatility and in how they interact with the columns stationary phase

• A detector records a signal as compounds exit the column, producing a chromatogram

Inside the column

Where does separation of GC primarily occur?

Inlet

What part of GC introduces a sample?

Helium (He), Hydrogen (H₂), Nitrogen (N₂)

Label the carrier gases…

1. ___: Most common in forensic labs; excellent chromatographic performance

2. ___: Faster separations possible; is flammable and requires safety considerations

3. ___: Lower cost, slower separations but suitable for some applications

Stationary phase

The thin coating on a GC capillary columns is the …

Stationary phase

What phase in GC affects which compounds interact with it and how well a mixture separates?

Isothermal, temperature-programmed

Label the two types of ways to run the GC oven…

1. ____: Oven held at a single consistent temperature throughout the entire run. Simpler but may not be ideal for mixtures with wide volatility ranges.

• Lower temperature → may separate early-eluting compounds well. but strongly retained compounds may take too long to exit

• Higher temperature → may shorten the run, but some compounds may exit too quickly to separate well

2. ____: Oven temperatures increases during the analysis. The rising temperature helps strongly retained compounds exit within a practical time.

• Lower temperature → helps retain and separate compounds that would otherwise exit quickly

• Higher temperature → helps more strongly retained compounds exit within a practical time

Volatility, column chemistry, stationary-phase interactions, oven temperature, and carrier gas flow

What 5 factors affect retention in GC?

Thermally stable and volatile

What types of compounds are suitable for GC?

Derivatization

What is a chemical reaction that modifies an analyte before analysis, producing a derivative with properties better suited for the method? It improves peak shape, volatility, thermal stability, and detection.

LC

What chromatography technique does this describe?

• Analytes must dissolve adequately and remain stable under the preparation and analysis conditions.

• Many drugs and polar metabolites can be analyzed using an appropriate method.

Pump, small stationary

Fill in the blanks:

Modern LC systems use a ____ to push the liquid mobile phase through a column packed with ____ ________ phase particles. The packed column resists flow, so the pump must generate pressure.

High-performance LC (HPLC), Ultra-hight performance LC (UHPLC), Reverse-phase LC (RP-LC)

Label the types of modern LC systems…

1. ____: Delivers mobile phase at a controlled flow rate through a packed column. It provides reproducible separations for many drugs and metabolites.

2. ____: Commonly used for smaller stationary-phase particles and equipment designed for higher pressures. It can provide faster analysis or improved separation compared with conventional HPLC. The benefit depends on the column and method conditions.

3. ____: The stationary phase is relatively non-polar and hydrophobic. Hydrophobic compounds generally interact more strongly with this phase. The mobile phase is commonly a mixture of water and an organic solvent. More hydrophobic compounds tend to remain in the column longer, while hydrophilic compounds exit sooner.

mobile-phase reservoirs, injector/autosampler, detector, high-pressure pump, column, and computer

What are the 6 major parts of an LC?

Mobile

What phase in LC transports the sample and influents separation?

Water, methanol, and acetonitrile

What are 3 common mobile phase components in LC?

Hydrophillic, hydrophobic

In reverse-phase LC, what compounds favor the mobile phase and spend more time traveling with the solvent (aka exit sooner)? What compounds interact better with the stationary phase and remain in the column longer (aka exit later)?

Isocratic, gradient

Label the types of elution in LC…

1. ____: The mobile phase composition stays the same throughout the separation. Works well when sample components have a similar range of retention.

2. ____: The mobile phase composition changes during the run. In RP-LC, the proportion of organic solvent usually increases. Increasing organic solvent makes the mobile phase stronger, helping strongly retained compounds elute sooner. Useful for mixtures containing compounds with a wide range of retention.

Stationary-phase interactions, solvent composition, analyte ionization, pH, and flow

What 5 factors affect retention in LC?

Stationary phase

In LC, the material bonded to particles packed inside a column is the …