GAS & HPLC Chromatography.

Chromatography: The separation of compounds in a mixture

GC/ Gas chromatography:

(“GCSE Chem Chromatography Practical | the Chemistry Blog”) 2016

Gas chromatography (GC) is an essential technique in

analytical chemistry, widely used for the separation and
analysis of compounds that can be vaporized without
decomposing. This method is commonly employed to assess
the purity of various substances or to isolate different
components within a mixture. Additionally, in preparative
chromatography, GC assists in obtaining pure compounds from
complex mixtures.
The process of gas chromatography involves injecting a
gaseous or liquid sample into a moving stream known as the
mobile phase, which is typically a carrier gas. This gas can be
helium, argon, nitrogen, or hydrogen, all of which are inert and
unreactive. The sample then passes through a stationary
phase, which can take the form of either a solid or a liquid.
Nowadays, most GC systems utilize a liquid stationary phase
made from polymers, housed within a separation column.

Temp Pressure

The carrier gas (He/N) is inserted into the tube which acts as a
mobile phase carried throughout the column. The temperature
and pressure of the gas is controlled which changes the rate of
gas flow. Pressure is usually kept at 5psi-10psi.
Contemporary GC columns are
generally made of fused silica and have
an inner diameter ranging from 100 to
320 micrometres (0.0039 to 0.0126
inches)-as thin as a strand of hair- and
can extend from 5 to 60 metres (16 to
197 feet) in length. Inside the column
are many small particles made up of
silica gel which is what acts as the
stationary phase in Gas
Chromatography. These columns are placed inside an oven that
allows for temperature control of the gas as it travels through.
the pressure is also controlled to change the flow of gas. The
effluent produced at the end of the column is continuously
monitored using an appropriate detector, ensuring precise
analysis of the compounds present in the sample.
Temperature and pressure of the gas will then separate the
mixture of: ethanol, propanol and butan-1-ol. This is what is
mixed and injected into the column with a hyponelic needle
(creating vapour).

According to the size of the chain, a smaller chain will come out
of the column first such as ethanol. However bigger chains like
butan-1-ol are heavier, slower molecules which will come out
the column last. This is called Retention time (how long
components spend inside the column).

HPLC/ High Performance Liquid Chromatography:

(“Weshine Liquid Chromatography”) 2014

High-Performance Liquid Chromatography, or HPLC, represents a

sophisticated technique within the realm of liquid chromatography. This
method is essential for the separation, identification, and quantification
of various components found in a mixture. HPLC finds extensive
application across multiple fields, including pharmaceuticals, food
science, environmental testing, and biochemical research. Its versatility
and precision make it a critical tool for scientists and researchers
working to analyse complex samples.
Despite GC and HPLC being two essential analytical techniques widely
utilized in the separation and analysis of various compounds; they differ
by their use of mobile phases. GC employs a gaseous mobile phase,
typically utilizing a carrier gas such as helium or nitrogen, which makes it
particularly effective for analysing volatile and thermally stable
compounds. On the other hand, HPLC utilizes a liquid mobile phase,
positioning it as the method of choice for non-volatile and thermally
unstable compounds, as well as larger molecules such as proteins and
pharmaceuticals. The design of the columns used in these techniques
also differs significantly; GC generally utilizes capillary columns lined
with a stationary phase, while HPLC relies on packed columns
containing solid stationary phases. In addition, GC relies on detectors
such as mass spectrometry whereas HPLC does not.

Using qualitative and quantitative GC and HPLC data to identify the

identity and amount of analytes (caffeine).

In qualitative analysis, techniques such as gas chromatography (GC)

and high-performance liquid chromatography (HPLC) play a vital role in
identifying compounds like caffeine through the comparison of retention
times. When a sample is introduced into the chromatographic system,
each component engages with the stationary phase in unique ways,
leading to their elution at distinct times. By analysing a known standard
of caffeine under identical conditions, researchers can determine a
characteristic retention time, which acts much like a fingerprint for that
compound. Should a peak corresponding to caffeine emerge in the
sample at this specific retention time, it provides strong evidence of
caffeine's presence. This retention time matching is essential for
establishing the identities of analytes in complex mixtures.
 On the quantitative side, these chromatographic methods also enable
the precise measurement of caffeine content by evaluating the area
under the peak associated with the analyte. The area of the peak is
directly related to the concentration of caffeine found in the sample. To
achieve accurate quantification of caffeine levels, analysts create
calibration curves using standards with known concentrations. By
comparing the peak area from the sample to the established calibration
curve, they can calculate the exact concentration of caffeine present. In
summary, while qualitative analysis confirms the identity of caffeine
through retention time matching, quantitative analysis focuses on
measuring the amount by integrating the calibrated peak areas.

Method for finding the amount of a given organic compound:

Developing a High-Performance Liquid Chromatography (HPLC) method
to quantify an organic compound in a sample entails several important
steps that require careful consideration. Initially, it is crucial to
understand the chemical and physical properties of the compound in
question. This includes evaluating its polarity, solubility, and stability, as
well as identifying any potential interferences that may arise from other
components in the sample matrix.
1. The following step is to choose the appropriate HPLC mode.
Reversed-phase HPLC (RP-HPLC) is the most used technique for
non-polar to moderately polar compounds; however, other modes
such as normal-phase, ion-exchange, or size-exclusion HPLC may
be more suitable depending on the specific characteristics of the
compound being analysed. A vital aspect of method development
is selecting an appropriate mobile phase. Usually, solvent systems
like water-methanol or water-acetonitrile are employed, and
adjustments may involve tuning the pH or incorporating additives
like ion-pairing agents when necessary. Additionally, the choice of
stationary phase is critical; for reversed-phase HPLC, a C18
column is typically favoured, considering factors like particle size
and column dimensions.
2. Once the column and mobile phase are established, it’s important
to optimize HPLC parameters. This entails setting the right flow
 rate (typically between 0.5 and 1.5 mL/min), injection volume
(usually between 5 and 50 µL), and temperature (generally at 25–
40°C). Selecting a suitable detection method, which could include
UV-Vis, fluorescence, or mass spectrometry, is also an integral
part of this optimization process.
3. Following the optimization, method validation is essential. This
step ensures that the HPLC procedure is both reliable and
reproducible. Validation involves assessing parameters such as
linearity, accuracy, precision, limits of detection (LOD) and
quantification (LOQ), along with selectivity and robustness.
Calibration is performed by preparing standard solutions and
constructing a calibration curve that correlates concentration to
peak area—internal standards may also be used when necessary.
4. Sample preparation is a critical phase in the process. This often
involves techniques such as filtration or centrifugation to eliminate
particulates, as well as liquid-liquid or solid-phase extraction (SPE)
to isolate the analyte of interest. Depending on the concentration
of the compound, further dilution or concentration may be required
prior to analysis. Eventually, the prepared sample is injected into
the HPLC system, and chromatograms are generated for
subsequent data analysis. The concentration of the organic
compound is determined by comparing the peak areas with the
calibration curve established earlier.
5. To ensure accuracy and robustness throughout the process,
method refinement and validation should be conducted under
varying conditions. By adhering to this structured approach, one
can develop a reliable, sensitive, and accurate HPLC method for
quantifying organic compounds in a given sample.

Conditions Ethanol Propan-1-ol Butan-1-ol Mixtures

200℃ 5Psi 0.85 0.91 1.02 0.85 0.91 1.01

200℃ 10Psi 0.80 1.05 1.59 0.80 1.05 1.59
100℃ 5Psi 0.74 0.80 0.87 0.74 0.80 0.97
100℃ 10 Psi 0.91 1.22 1.81 0.91 1.22 1.81

Optimized separation from an instrumental chromatographic technique

Optimizing separation in chromatographic techniques, such as Gas
Chromatography (GC) and High-Performance Liquid Chromatography (HPLC),
involves carefully managing various parameters to improve resolution,
efficiency, and sensitivity.
In the case of GC, the type of column plays a crucial role in the separation
process. Key factors include the column's length, internal diameter, and the
composition of the stationary phase. Typically, longer columns or those with
smaller internal diameters enhance separation effectiveness by increasing the
number of theoretical plates available for the separation process. Selecting the
right stationary phase is essential and should be based on the analyte's polarity,
facilitating suitable interactions for effective partitioning of the components.
Temperature programming is another important aspect; employing a
temperature gradient can significantly improve separation by allowing different
volatile compounds to elute at varying rates, which enhances peak resolution
and reduces overall analysis time. Additionally, the choice of carrier gas (such
as helium, nitrogen, or hydrogen), along with its flow rate and injection
technique (either split or split-less), can markedly influence the sharpness of
peaks and their corresponding retention times.
Similarly, in HPLC, the optimization of separation relies on the careful
selection of the mobile phase, stationary phase, and operating conditions. The
type of chromatography—be it reversed-phase, normal-phase, ion-exchange, or
size-exclusion—should align with the chemical characteristics of the analyte.
For instance, in reversed-phase HPLC, a C18 column is commonly utilized, and
the mobile phase might consist of specific mixtures, like water-methanol or
water-acetonitrile, which are adjusted to achieve optimal resolution. The pH of
the mobile phase is also a significant factor that governs the ionization of
analytes, impacting their retention times and the shapes of their corresponding
peaks. Additionally, to strike a balance between analysis time and resolution,
the flow rate, column temperature, and gradient elution technique should be
fine-tuned. Gradient elution, where the composition of the mobile phase
changes over time, can markedly enhance separation efficiency, especially in
the analysis of complex mixtures. Moreover, aspects such as injection volume,
sample preparation, and detector settings also play a critical role in determining
the shape of peaks and the overall performance of the chromatographic system.
By appropriately selecting and optimizing these parameters for both GC and
HPLC, analysts can achieve high-resolution separation, better peak symmetry,
and improved detection of target analytes. This leads to more accurate and
reliable analytical results, which are paramount in various scientific and
industrial applications.

References:
“GCSE Chem Chromatography Practical | the Chemistry Blog.” ReAgent Chemical Services,

24 Jan. 2024, www.chemicals.co.uk/blog/gcse-chemistry-practical-chromatography.

Wikipedia Contributors. “Gas Chromatography.” Wikipedia, Wikimedia Foundation, 16 Mar.

2019, en.wikipedia.org/wiki/Gas_chromatography.

Aryal, Sagar. “High-Performance Liquid Chromatography (HPLC).” Microbiology Notes, 24

May 2024, microbenotes.com/high-performance-liquid-chromatography-hplc/.

Chromatography Today. “Understanding the Difference between Retention Time and

Relative Retention Time.” Chromatography Today, Chromatography, 2019,

www.chromatographytoday.com/news/autosamplers/36/breaking-news/understanding

-the-difference-between-retention-time-and-relative-retention-time/31166.