Chromatography Techniques — MCQs
Which of the following separates proteins solely on the basis of their molecular size?
Which method is used to separate a mixture of lipids?
The most specific test to detect blood stains is:
Glycated hemoglobin (HbA1c) is best measured using?
The technique shown in the image is:
Protein purification and separation can be done by all except:
Which technique is used for protein separation based on molecular size?
Expression and release of a repressed emotion is called as
Which of the following methods of protein separation is not dependent on molecular size?
The shown pattern in electrophoresis is due to:
Chromatography Techniques Indian Medical PG Practice Questions and MCQs
Question 1: Which of the following separates proteins solely on the basis of their molecular size?
- A. Isoelectric focusing
- B. Chromatography on a diethylaminoethyl (DEAE) cellulose column
- C. Gel filtration chromatography (Correct Answer)
- D. Chromatography on a carboxymethyl (CM) cellulose column
Explanation: ***Gel filtration chromatography*** - Also known as **size-exclusion chromatography**, this method separates proteins based on their **hydrodynamic volume** (molecular size and shape). - Larger proteins pass through the column more quickly because they are excluded from the pores of the stationary phase, while smaller proteins enter the pores and have a longer, more tortuous path. *Isoelectric focusing* - This technique separates proteins based on their **isoelectric point (pI)**, which is the pH at which the protein has no net electrical charge. - Proteins migrate through a pH gradient until they reach the point where their net charge is zero. *Chromatography on a diethylaminoethyl (DEAE) cellulose column* - **DEAE cellulose** is an **anion-exchange resin**, meaning it binds **negatively charged** proteins. - Separation is based on the **net charge** of the protein at a given pH. *Chromatography on a carboxymethyl (CM) cellulose column* - **CM cellulose** is a **cation-exchange resin**, meaning it binds **positively charged** proteins. - Separation is based on the **net charge** of the protein at a given pH.
Question 2: Which method is used to separate a mixture of lipids?
- A. Electrophoresis
- B. Chromatography (Correct Answer)
- C. Isoelectric focusing
- D. PAGE
Explanation: ***Chromatography*** - **Chromatography** (e.g., thin-layer chromatography, gas chromatography, high-performance liquid chromatography) is widely used to separate lipids based on differences in their **polarity**, **molecular weight**, or **solubility** in various solvents. - This method allows for the isolation and identification of different lipid classes and individual lipid species from a complex mixture. *Electrophoresis* - **Electrophoresis** separates molecules based on their **charge** and **size** in an electric field, making it more commonly used for proteins and nucleic acids. - Lipids are generally **uncharged** or have very low charge, which makes them poorly suited for separation by standard electrophoretic methods without modification. *Isoelectric focusing* - **Isoelectric focusing** is a type of electrophoresis that separates molecules based on their **isoelectric point (pI)**, which is the pH at which a molecule has no net charge. - This technique is primarily used for **proteins** and **peptides**, as lipids typically lack ionizable groups necessary for establishing a distinct pI. *PAGE* - **PAGE** (Polyacrylamide Gel Electrophoresis) is a common method used to separate **proteins** and **nucleic acids** based on their size and charge. - Lipids are **hydrophobic** and do not readily migrate through an aqueous polyacrylamide gel matrix, making PAGE unsuitable for their direct separation.
Question 3: The most specific test to detect blood stains is:
- A. Benzidine test
- B. Teichmann's test
- C. Spectroscopic test (Correct Answer)
- D. Orthotoluidine test
Explanation: ***Spectroscopic test*** - The **spectroscopic test** is considered the most specific for detecting blood stains because it identifies the characteristic absorption bands of **hemoglobin** and its derivatives. - This test is highly definitive due to the unique **light absorption properties** of blood components, making it less prone to false positives compared to chemical tests. *Benzidine test* - The **benzidine test** is a sensitive preliminary test for blood but is **not specific**, as it reacts with other oxidizing agents (e.g., rust, certain plant peroxidases). - It works by detecting the **peroxidase-like activity of hemoglobin**, leading to color changes but lacks confirmation of blood origin. *Teichmann's test* - **Teichmann's test** (hemin crystal test) is a moderately specific confirmatory test that produces **rhombic crystals of hemin** when heated with glacial acetic acid and a halide salt. - While more specific than presumptive tests, it can sometimes produce **false-negative results** with old or degraded bloodstains and may be less sensitive than spectroscopy. *Orthotoluidine test* - Similar to the benzidine test, the **orthotoluidine test** is another **presumptive test** that detects the peroxidase-like activity of hemoglobin, resulting in a blue-green color change. - It is **highly sensitive but not specific**, meaning it can also give positive reactions with other substances that have similar peroxidase activity, leading to potential false positives.
Question 4: Glycated hemoglobin (HbA1c) is best measured using?
- A. Isoelectric focusing
- B. Affinity chromatography
- C. Electrophoresis
- D. Ion exchange chromatography (Correct Answer)
Explanation: ***Ion exchange chromatography*** - This method separates hemoglobin variants based on their **charge differences** due to the glucose molecule attached to HbA1c. - It is a highly sensitive and specific method for quantifying HbA1c, widely used in clinical laboratories. *Isoelectric focusing* - This technique separates molecules based on their **isoelectric point (pI)**, the pH at which they have no net charge. - While it can differentiate some hemoglobin variants, it is generally **less efficient and more complex** for routine HbA1c measurement compared to ion exchange chromatography. *Affinity chromatography* - This method separates molecules based on their **specific binding affinity** to a ligand immobilized on a stationary phase. - While it has been explored for HbA1c measurement, it is **not the most commonly used** or preferred method due to potential interferences and cost compared to ion exchange chromatography. *Electrophoresis* - This technique separates molecules based on their **charge and size** in an electric field. - While it can separate major hemoglobin variants, it has **lower resolution and accuracy** for routine HbA1c quantification compared to more specialized chromatographic methods, making it less ideal for precise measurement.
Question 5: The technique shown in the image is:
- A. High performance liquid chromatography (Correct Answer)
- B. Haemoglobin electrophoresis
- C. Gel electrophoresis
- D. Tandem mass spectrometry
Explanation: ***High performance liquid chromatography*** - The image displays a **chromatogram** with distinct peaks labeled HbA1c, HbF, HbA0, and HbA2, separated based on their chemical properties. - This separation and detection method is characteristic of **High Performance Liquid Chromatography (HPLC)**, a technique used for quantifying different hemoglobin fractions. *Tandem mass spectrometry* - **Tandem mass spectrometry (MS/MS)** identifies compounds based on their mass-to-charge ratio and fragmentation patterns, which would look like mass spectra, not peaks on a time-based chromatogram. - While MS/MS is highly sensitive and specific, it doesn't produce the type of **elution profile** seen in the image. *Haemoglobin electrophoresis* - **Hemoglobin electrophoresis** separates hemoglobins based on their electrical charge, resulting in bands on a gel or a densitometric scan, not the **distinct chromatogram peaks** shown here. - While used for hemoglobin analysis, the visual representation is typically different, often displaying bands that reflect migration distance. *Gel electrophoresis* - **Gel electrophoresis** separates molecules, such as proteins or nucleic acids, by size and charge through a gel matrix, producing distinct **bands** that can be visualized. - This method would not produce the continuous **elution peaks over time** as observed in the provided graph, which indicates a liquid chromatography technique.
Question 6: Protein purification and separation can be done by all except:
- A. Densitometry (Correct Answer)
- B. Electrophoresis
- C. Centrifugation
- D. Chromatography
Explanation: ***Correct: Densitometry*** - **Densitometry** is a quantification technique used to measure the optical density or darkness of a material - It quantifies the amount of protein in a gel or blot **after separation has already occurred** by other methods - It does **not separate or purify** proteins; it only measures and quantifies them - Used for analyzing results from electrophoresis, Western blots, and similar techniques *Incorrect: Electrophoresis* - Separates proteins based on **charge, size, or shape** when subjected to an electric field through a matrix - Common techniques include SDS-PAGE (by molecular weight), IEF (by isoelectric point), and native PAGE - Routinely used for both analytical separation and preparative purification of proteins *Incorrect: Centrifugation* - Separates proteins based on **molecular weight, shape, and density** using centrifugal forces - Used for initial clarification of cell lysates, separating organelles, and concentrating proteins - Differential and density gradient centrifugation are key purification techniques *Incorrect: Chromatography* - Encompasses multiple techniques: **ion-exchange** (charge), **size exclusion** (molecular weight), **affinity** (specific binding), and **hydrophobic interaction** chromatography - Highly effective for both analytical separation and purification of proteins to high purity - Gold standard for protein purification in biochemistry laboratories
Question 7: Which technique is used for protein separation based on molecular size?
- A. Affinity chromatography
- B. Gel filtration chromatography (Correct Answer)
- C. HPLC
- D. Salting out
Explanation: ***Gel filtration chromatography*** - Also known as **size-exclusion chromatography**, this method separates proteins by passing them through a porous matrix. **Larger proteins** elute first as they cannot enter the pores, while smaller proteins get trapped and elute later. - This technique effectively separates proteins based solely on their **hydrodynamic radius**, which is closely related to their molecular size. *Affinity chromatography* - This method separates proteins based on their **specific binding affinity** to a ligand immobilized on a stationary phase, not molecular size. - It is used for purifying proteins that bind to a specific molecule, such as an antibody or substrate. *HPLC* - **High-performance liquid chromatography** is a general technique that can use various separation mechanisms (e.g., reverse-phase, ion-exchange, size-exclusion) under high pressure. - While it *can* be used for size-exclusion, HPLC itself describes the *method* of chromatographic performance rather than a specific separation principle based on molecular size alone. *Salting out* - This technique separates proteins based on their **solubility** in high salt concentrations. - As salt concentration increases, the proteins lose their hydration shells and precipitate out of solution, with different proteins precipitating at different salt concentrations.
Question 8: Expression and release of a repressed emotion is called as
- A. Dissociation
- B. Confabulation
- C. Abreaction (Correct Answer)
- D. Regression
Explanation: **Abreaction** - This term refers to the **expression and release of a repressed emotion**, often in a therapeutic context, providing psychological relief. - It involves re-experiencing a traumatic event or repressed emotions to alleviate their negative impact. - Also known as **catharsis** in psychoanalytic therapy. *Dissociation* - **Dissociation** involves a disruption in the normal integration of consciousness, memory, identity, emotion, perception, body representation, motor control, and behavior. - It describes a mental process that causes a lack of connection in a person's thoughts, memories, feelings, actions, or sense of identity, rather than an active release of emotion. *Confabulation* - **Confabulation** is the creation of false memories in the absence of an intention to deceive. - It is often seen in individuals with specific neurological or psychiatric conditions, where they unconsciously fill in gaps in their memory with fabricated details. *Regression* - **Regression** is a defense mechanism in which an individual faced with anxiety or stress retreats to an earlier developmental stage. - It involves reverting to immature patterns of behavior, rather than the release of a specific, repressed emotion.
Question 9: Which of the following methods of protein separation is not dependent on molecular size?
- A. Gel filtration chromatography
- B. SDS-PAGE
- C. Ultracentrifugation
- D. Ion-exchange chromatography (Correct Answer)
Explanation: ***Ion-exchange chromatography*** - This method separates proteins based on their **net charge** at a specific pH. - Proteins bind to a charged resin based on their charge, independent of their size. *Gel filtration chromatography* - Separates proteins based on their **molecular size** and shape as they pass through a porous matrix. - **Larger molecules** elute first as they cannot enter the pores, while smaller molecules are retained. *SDS-PAGE* - **Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis** separates proteins primarily based on their **molecular weight**. - SDS denatures proteins and confers a uniform negative charge, allowing migration through a gel matrix based on size. *Ultracentrifugation* - Separates macromolecules and particles based on their **sedimentation rate**, which is influenced by **molecular mass**, density, and shape. - While molecular size is a factor, density and shape also play significant roles in the separation process.
Question 10: The shown pattern in electrophoresis is due to:
- A. Charges (Correct Answer)
- B. Molecular weight
- C. Bound oxygen
- D. Hydrophobicity
Explanation: ***Charges*** - Electrophoresis separates molecules, such as **hemoglobin variants**, based on their **electrical charge** when placed in an electric field. - Different amino acid substitutions in hemoglobin lead to changes in net charge, causing them to migrate at different rates in the gel, as seen by the distinct bands for Hb A, Hb S/D, and Hb A2. *Molecular weight* - While molecular weight can influence migration in some electrophoretic techniques (e.g., SDS-PAGE), standard hemoglobin electrophoresis primarily separates based on **charge differences**, not molecular size. - All common hemoglobin variants have a very similar **molecular weight** (approximately 64,500 Da), so this factor would not effectively separate them into distinct bands. *Bound oxygen* - The amount of **bound oxygen** to hemoglobin does not significantly alter its overall electrical charge or molecular weight to cause distinct bands in electrophoresis. - Oxygen binding is a dynamic process and does not account for the **structural differences** in hemoglobin variants that lead to their electrophoretic separation. *Hydrophobicity* - Hydrophobicity is a characteristic of molecules, but it is not the primary principle by which standard **gel electrophoresis** separates hemoglobin variants. - Techniques like **hydrophobic interaction chromatography** would exploit hydrophobicity, not the electrophoretic gel shown.
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