Analytical Chemistry PDF

# Mode Analytical Chemistry ## 12A Chromatographic Separations - The process of separating a mixture of compounds is called **chromatography**. - The process is based on the differential distribution of the analytes between a stationary phase and a continuously moving mobile phase. - In the **stat...

# Mode Analytical Chemistry ## 12A Chromatographic Separations - The process of separating a mixture of compounds is called **chromatography**. - The process is based on the differential distribution of the analytes between a stationary phase and a continuously moving mobile phase. - In the **stationary phase**, the molecules are retained by a specific interaction. - In the **mobile phase**, the molecules are transported throughout the system. - The process was developed by **Mikhail Tsvet** in 1901 to separate pigments from plant extracts. - The stationary phase was a glass column packed with solid adsorbent material and the mobile phase was a liquid solvent. - The most frequently used type of chromatography is **liquid chromatography**, in which the mobile phase is a liquid. - **Gas chromatography** is used to separate volatile substances. - **Other types of chromatography** include **ion-exchange chromatography, size-exclusion chromatography, and affinity chromatography**. ## 12A.1 General Theory of Chromatography - *Chromatographic separations are based on the distribution of analytes between a stationary phase, which is a solid or liquid that is fixed to a support, and a flowing mobile phase, which is a gas or a liquid.* - The stationary phase is chosen based on the types of interactions that are needed to separate the analytes, such as **adsorption, partitioning, ion exchange, size exclusion, or affinity**. - The mobile phase is chosen to complement the stationary phase and allow for the separation of the analytes. - The choice of stationary phase and mobile phase is critical to a successful chromatographic separation. ## 12A.2 Classifying Analytical Separations - Analytical separations can be classified based on: - The physical state of the mobile and stationary phases - The type of interaction between the mobile and stationary phases - The chemical or physical mechanism responsible for separating the mixture. - The mobile phase is **always either a gas or a liquid**. - The stationary phase is either a **solid or a liquid**: - **In adsorption chromatography**, the stationary phase is a solid, and the solutes are attracted to the surface of the stationary phase by electrostatic interactions. - **In partition chromatography**, the stationary phase is a liquid, and the solutes distribute themselves between the two phases based on their relative solubilities. - **In ion-exchange chromatography**, the stationary phase contains ionic functional groups that attract oppositely charged solutes. - **In size-exclusion chromatography**, the stationary phase is a porous material that separates solutes based on their size. - The **affinity of the analyte for the stationary phase** will determine its **retention time** on the chromatographic column. ## 12A.3 General Theory of Column Chromatography - **Column chromatography** is a technique for separating a mixture of compounds based on their differential distribution between the mobile phase and the stationary phase. - ***The sample is introduced at the top of the column, and the different analytes are separated as they migrate through the column at different rates.*** - ***The rate at which an analyte moves through the column depends on its affinity for the stationary phase, with analytes that have a higher affinity for the stationary phase being retained longer and moving more slowly.*** - At the end of the column, a detector is used to measure the concentration of the eluted analytes. - The separation is monitored with a suitable detector situated at the end of the column. - A **chromatogram** is a plot of the detector's signal as a function of time or volume of eluted mobile phase. - Each **chromatographic peak** corresponds to a single analyte. - The **retention time**, $t_r$, is the time it takes for a solute to move from its point of injection to the peak maximum. - The **retention volume**, $V_r$, is the volume of mobile phase needed to move the solute from its point of injection to the detector. - The **baseline width**, $w$, is the width of a solute's peak at the baseline. ## 12B. 1 Chromatographic Resolution - **Resolution** is a quantitative measure of the **separation between two chromatographic peaks**, A and B. - $R = \frac{2(t_{r,B}-t_{r,A})}{w_B+w_A}$ - The **separation** between two chromatographic bands improves with an increase in $R$. - For two peaks of equal size, a **resolution** of 1.5 corresponds to an overlap in area of only 0.13%. - **Resolution** provides a method to determine if a change in experimental conditions leads to increased separation. ## 12B.2 Capacity Factor - The distribution of a solute, S, between the mobile phase and stationary phase can be represented by: - Equilibrium reaction: $S_m \rightleftharpoons S_s$ - Partition coefficient: $K_D = \frac{[S_s]}{[S_m]}$ - Distribution ratio: $D = \frac{[S_s]_{tot}}{[S_m]_{tot}}$ - The **capacity factor**, $k'$, is a measure of how strongly a solute is retained by the stationary phase. - $k' = DV_m/V_s$ - $k' = \frac{t_r - t_m}{t_m}$ - The **adjusted retention time**, $t'$, is the time it takes for a solute to elute from the column after the void volume has been eluted ($t' = t_r - t_m$). ## 12B.3 Column Selectivity - The relative selectivity of a chromatographic column for a pair of solutes is given by the **selectivity factor**, $\alpha$. - $\alpha = \frac{k'_B}{k'_A} = \frac{t_{r,B}-t_m}{t_{r,A}-t_m}$ - The **selectivity factor** is a measure of the relative affinity of the column for the two solutes. - The selectivity factor is equal to 1 when the solutes elute with identical retention times. - The selectivity factor is greater than 1 when $t_{r,B}$ is greater than $t_{r,A}$. ## 12B.4 Column Efficiency - **Column efficiency** provides a quantitative measure of the extent of **band broadening**, the process in which a solute's baseline width continually increases as it moves through the column. - **Column efficiency** is defined in terms of: - **The number of theoretical plates**, N, or - **The height of a theoretical plate**, H. - $N = \frac{L}{H}$ - **A column's efficiency improves with an increase in the number of theoretical plates or a decrease in the height of a theoretical plate.** - $N = 16 \frac{t_r}{w}$ - $N = 5.545 \frac{t_r}{w_{1/2}}$ ## 12B.5 Peak Capacity - **Peak capacity**, $n_c$, is the maximum number of solutes that can be baseline resolved on a given column. - $n_c = 1+\frac{4}{\sqrt{N}}\frac{V_{max}}{V_{min}}ln\frac{V_{max}}{V_{min}}$ - The **peak capacity** provides an estimate of the number of solutes that can be separated on a given column. - The **peak capacity** increases with an increase in the number of theoretical plates and a decrease in the volume of mobile phase required to elute the solutes. ## 12B.6 Nonideal Behavior - Ideal chromatographic behavior occurs when the **solute's partition coefficient, KD, is constant for all concentrations of solute**. - In some cases, **chromatographic peaks** show **nonideal behavior**, leading to **asymmetrical peaks**, which can be either **fronted** (a tail at the **beginning** of the peak) or **tailed** (a tail at the **end** of the peak). - **Fronting** occurs when the column is **overloaded** with the sample. - **Tailing** occurs when some sites on the stationary phase retain the solutes more strongly than other sites. ## 12C Optimizing Chromatographic Separations - The **resolution** between two chromatographic peaks can be optimized by adjusting the: - Capacity factor, $k'$ (adjusting the affinity of the analyte for the stationary phase) - Selectivity factor, $\alpha$ (adjusting the relative affinity of the column for the two solutes) - Number of theoretical plates, N (adjusting the efficiency of the column). - $R = \frac{1}{ \sqrt(N)}(\frac{ \alpha - 1}{ \alpha })(\frac{ k_B}{1+k_B})$ - Increasing $k'$ generally improves resolution, but the **effect is less pronounced as k' increases**. - Increasing k' also **increases the retention time**. - To change $k'$ without significantly changing $\alpha$, adjust chromatographic conditions in a way that leads to a **nonselective increase in the capacity factor for both solutes** by: - **Decreasing the temperature for gas phase.** - **Changing the mobile phase for liquid phase.** - Adjusting the **selectivity factor, $\alpha$**, generally has a more dramatic effect on resolution than adjusting $k'$. - To change $\alpha$: - **Vary the pH for weak acids or weak bases.** - **Change the composition of the mobile phase.** - To increase the **number of theoretical plates**, N, either **increase the length of the column** or **decrease the height of a theoretical plate**, H. - **Decreasing particle size in packed columns** decreases the contribution of multiple paths (Hp) and mass transfer (Hs and Hm to the height of a theoretical plate, H. - **Open tubular columns** (capillary columns) do not contain a particulate packing material, which reduces the contributions of multiple paths and mass transfer to H. - **Capillary columns** typically provide a better separation efficiency than **packed columns** because of their longer lengths and smaller internal diameters (less band broadening) but they can only handle smaller samples. ## 12C.1 Using the Capacity Factor to Optimize Resolution - Increasing $k'$ generally improves resolution, but the **effect is less pronounced as k' increases**. - Increasing $k_B$ also **increases the retention time**, which can be undesirable for a faster analysis. - Adjusting the **capacity factor** for solute B is an **easy yet effective way** to improve resolution. ## 12C.2 Using Column Selectivity to Optimize Resolution - Adjusting the **selectivity factor, $\alpha$**, often has the **most dramatic effect** on resolution. - To change $\alpha$ in **liquid phase**, the common approach is to change one or more of the mobile-phase solvents. - In **reverse-phase chromatography**, changing the composition of the mobile phase commonly includes: - **Acetonitrile** - **Methanol** - **Tetrahydrofuran** (THF) - **Using a solvent triangle** in a reverse-phase separation can help **optimize the mobile phase** and produce the best separation in a reasonable amount of time. - To change $\alpha$ in **gas phase**: - **Alter the stationary phase** ## 12C.3 Using Column Efficiency to Optimize Resolution - **Column efficiency** is a measure of the extent of **band broadening**, the process in which a solute's baseline width continually increases as it moves through the column. - To increase the **number of theoretical plates**, N, either **increase the length of the column** or **decrease the height of a theoretical plate**, H, which is directly proportional to **band broadening**. - **Decreasing the particle size** in packed columns can decrease H. - **Open tubular columns** (capillary columns) do not contain a particulate packing material, which also decreases H. ## 12C.4 Using Temperature Programming or Gradient Elution to Optimize Resolution - **Temperature programming** and **gradient elution** are techniques that can **adjust the capacity factor** of the analytes over time, **optimizing the separation** of both early and late eluting components. - **In temperature programming** the column temperature is **increased** over time, which **shortens the retention time for later eluting components**. - **In gradient elution** the mobile phase's solvent strength is **increased** over time, which also **shortens the retention time for later eluting components**. ## 12D Gas Chromatography - **Gas chromatography**, GC, is a technique for separating a mixture of compounds based on their differential distribution between the mobile phase (a gas) and the stationary phase (a liquid or solid). - **The sample is injected into the GC as a gas or liquid**, then **vaporized** and carried through the column by an inert gas, which is called the **carrier gas**. - **The sample's components are separated based on their volatilities and affinities for the stationary phase.** - **The separated components are detected with a detector** at the end of the column. ## 12D.1 Mobile Phase - The most common carrier gases used in GC are **He, Ar, and N2**. - The carrier gas should be inert so that it does not react with the sample or the stationary phase. - **The flow rate of the carrier gas** depends on the type of column used. The flow rate is **higher for packed columns** and **lower for capillary columns**. ## 12D.2 Chromatographic Columns - **Packed columns** contain a particulate packing material that serves as a support for the stationary phase (a liquid). - **Capillary columns** are open tubular columns that do not contain a packing material. - **Capillary columns offer higher efficiency** and **lower pressure drops** than packed columns. ## 12D.3 Stationary Phases - The **stationary phase** in GC influences the selectivity of the separation. - **Nonpolar phases** are good for separating nonpolar analytes, while **polar phases** are better for separating polar analytes. - The most frequently used stationary phases in GC: - **Squalane:** Nonpolar - **Polydimethyl siloxane:** Slightly polar - **50% methyl-50% phenyl polysiloxane:** Moderately polar - **50% trifluoropropyl-50% methyl polysiloxane:** Moderately polar - **50% cyanopropyl-50% phenylmethyl polysiloxane:** Polar - **Polyethylene glycol:** Polar - **Bonded stationary phases** are more stable than **unbonded stationary phases** because they are chemically attached to the support material. ## 12D.4 Sample Introduction - **To be separated by GC, all analytes must be volatile.** - Nonvolatile analytes must be **converted to a volatile derivative** before analysis. - The **concentration of the analyte** should be appropriate for the detector, and the sample should not be overloaded. - **Sample injection** techniques include: - **Split injection**: In split injection, a small portion of the sample enters the column, while the rest is vented to waste. - **Splitless injection**: In splitless injection, a larger portion of the sample enters the column, increasing the sensitivity of the analysis. - **On-column injection**: In on-column injection, the sample is injected directly onto the column, which is useful for thermally unstable samples. ## 12D.5 Temperature Control - **The temperature of the GC column is critical to attaining a good separation.** - **Isothermal separations** maintain the column at a constant temperature, which is typically set below that of the lowest boiling analyte. - **Temperature programming:** The column temperature is increased over time, which can improve the separation of analytes with a wide range of boiling points. ## 12D.6 Detectors for Gas Chromatography - The common GC detectors include: - **The thermal conductivity detector**, TCD, is a universal detector that is sensitive to changes in the thermal conductivity of the carrier gas. - **The flame ionization detector**, FID, is nearly a universal detector that is sensitive to organic compounds. - **The electron capture detector**, ECD, is a selective detector that is sensitive towards halogen and nitro functional groups. - **The flame photometric detector** is selective for compounds containing phosphorus or sulfur. - **The thermionic detector** is sensitive to compounds containing nitrogen or phosphorus. - **The Fourier transform infrared spectrophotometer**, FT-IR, is a sensitive detector that provides a spectral fingerprint of the analyte. - **The mass spectrometer**, MS, is a sensitive detector that provides structural information about the analyte. ## 12D.7 Quantitative Applications - **GC is widely used for quantitative analyses** in various fields, including environmental monitoring, pharmaceuticals, food science, and forensic analysis. - The most common methods used in quantitative GC analysis include: - **External standard method**: A calibration curve is constructed by analyzing a series of external standards. - **Internal standard method**: An internal standard is added to the sample, and the analyte's concentration is determined relative to that of the internal standard. ## 12D.8 Qualitative Applications - **GC is used for identifying analytes** in complex mixtures. - Qualitative GC applications can be accomplished with a mass spectrometer, FT-IR, or Kovats retention indexes. - **Kovats retention indexes**, I, provide a means for normalizing retention times by comparing the retention time of a solute with those of normal alkanes. - $I_{cpd} = 100 \frac{( log \; t'_R)_{cpd} - ( log \; t'_R)_x}{(log \; t'_R)_{x+1} - (log \; t'_R)_x} + I_x$ ## 12E High-Performance Liquid Chromatography - **High-performance liquid chromatography**, HPLC, is a technique that separates a mixture of compounds based on their differential distribution between the mobile phase (a liquid) and the stationary phase (a solid or liquid). - HPLC is a powerful analytical technique that can be used to separate a wide variety of compounds that are not volatile or thermally unstable. - HPLC is commonly used in **pharmaceutical, environmental, and forensic analysis.** - The **stationary phase in HPLC** is typically coated on a **particulate packing material** that is contained within the **column**. - The most frequently used types of column chromatography in HPLC are: - **Normal-phase chromatography**: A polar stationary phase and a nonpolar mobile phase. - **Reverse-phase chromatography**: A nonpolar stationary phase and a polar mobile phase. - The **mobile phase** is typically composed of a mixture of solvents, and the **composition of the mobile phase can be varied to optimize the separation**. - **Gradient elution** is a technique where the **composition of the mobile phase is changed over time** during the separation, allowing for the separation of solutes with a wide range of polarities. - **HPLC detectors** are commonly based on **spectroscopic**, **electrochemical**, or **refractive index measurements**. ## 12E.1 HPLC Columns - **HPLC columns** are typically constructed from **stainless steel**, with internal diameters ranging from 2.1 mm to 4.6 mm. - The columns are packed with **3–10 µm porous silica particles**. - **Microcolumns** are smaller in internal diameter and packed with smaller particles, increasing efficiency and lowering solvent use. - **Open tubular columns** are even smaller than microcolumns, providing high resolution and efficiency, but are harder to manufacture. - **Guard columns** are placed before the **analytical columns** to protect them from contamination by particulate material or irreversible binding of solutes to the stationary phase. ## 12E.2 Stationary Phases - **In liquid-liquid chromatography**, a film of liquid stationary phase is coated on a particulate packing material. - **Bonded stationary phases** have superior stability because they are covalently bound to the support material. - **In normal-phase chromatography**, the stationary phase is polar, while the mobile phase is nonpolar and the analytes are separated according to their polarity, with the least polar eluting first. - **In reverse-phase chromatography**, the stationary phase is nonpolar, while the mobile phase is polar and the analytes are separated according to their polarity, with the most polar eluting first. ## 12E.3 Mobile Phases - **The mobile phase** is a liquid that is continuously pumped through the HPLC column. **The polarity of the mobile phase can be adjusted to optimize the separation**. - **The polarity of the mobile phase** is characterized by its **polarity index**, $P'$, with higher polarity indexes corresponding to polar solvents and lower polarity indexes corresponding to nonpolar solvents. - **A gradient elution** is a technique where the **composition of the mobile phase is changed over time**, allowing for the separation of solutes with a wide range of polarities. ## 12E.4 HPLC Plumbing - **HPLC systems** require several **solvent reservoirs**, pumps, a **pulse damper**, and a **proportioning valve** to deliver the mobile phase to the column. - **The solvents** need to be **degassed** and **filtered** before being used to prevent the formation of gas bubbles and potential clogging of the column. - **The pump** is the most critical component of an HPLC system, as it is responsible for delivering the mobile phase under **high pressure** and at a **constant flow rate**. - **The gradient elution** is accomplished by **changing the composition of the mobile phase over time**, which is often achieved with a **proportioning valve that controls the mixture of solvents**. ## 12E.5 Sample Introduction - **In HPLC, the samples are injected using a loop injector.** - The **loop injector** is a device where the sample is placed in a small loop of tubing that is then flushed with the mobile phase. ## 12E.6 Detectors for HPLC - The most common **HPLC detectors** are based on **spectroscopic**, **electrochemical**, or **refractive index measurements.** - **UV/Vis detectors**, are the most common and measure the absorbance of the analyte at a specific wavelength. - **Fluorescence detectors** measure the fluorescence intensity of the analyte at a specific wavelength and are more sensitive than UV/Vis detectors but fewer analytes are capable of fluorescing. - **Electrochemical detector**s measure the current that flows between two electrodes when the analyte is oxidized or reduced at a specific potential. - **Refractive index detectors** are the most sensitive, but they are often limited by the mobile phase's absorbance. - **Mass spectrometers** are versatile detectors that can measure the mass-to-charge ratio of the analyte, providing a unique identification. ## 12E.7 Quantitative Applications - **HPLC is widely used for quantitative analyses** in a variety of fields, including environmental monitoring, pharmaceuticals, food science, and forensic analysis. - **Quantitative HPLC analysis** is typically carried out using **external or internal standards**. - **External standards** are used to construct a calibration curve by analyzing a series of standards with known concentrations. - **Internal standards** are added to the sample, and the concentration of the analyte is determined relative to the concentration of the internal standard. ## 12E.8 Representative Method: Determination of Fluoxetine in Serum - **Fluoxetine** is the antidepressant drug known by the brand name Prozac. - The determination of **fluoxetine** in biological samples is a complex analysis that typically involves **sample preparation** (often a **solid-phase extraction**) followed by **HPLC separation** using a **fluorescence detector**. ## 12D.10 Evaluation - **Gas chromatography** is a powerful analytical technique that can be used to separate and quantify a wide variety of compounds. - The **advantages** of GC include: - High sensitivity - High resolution - Excellent selectivity - The **disadvantages** of GC include: - The need for the analyte to be volatile - The potential for sample degradation - The need for specialized equipment ## 12E.10 Evaluation - **HPLC** is a versatile technique that can be used to separate and quantify a wide variety of compounds, including those that are not volatile or thermally unstable. - The **advantages** of HPLC include: - Great sensitivity - Excellent resolution - The ability to separate a variety of compounds, including those that are not volatile or thermally unstable - The **disadvantages** of HPLC include: - Higher expense than gas chromatography - The potential for sample degradation - The need for specialized equipment # Conclusion - **Chromatography** is a versatile technique that is widely used in a variety of analytical fields. - **GC** is a powerful technique for separating volatile compounds, while **HPLC** is better suited for nonvolatile compounds. - The proper choice of **stationary phase, mobile phase, and operating conditions** is critical to obtaining a successful chromatographic separation. - **The development of new techniques** and **instrumentation** is constantly improving the capabilities and applications of chromatography. - **The future of chromatography** is bright, as it will continue to play a critical role in many aspects of science and engineering.