What causes Shifting retention time?
- Mobile phases
- Temperature shifting or wrong in method
- Solvent selection valve leaking so gradient off. Use A2/B2 or can bypass valve altogether
- Problem with the mixer
- Leak from inside - as in leaking from inside into each other
What are 4 Factors Affecting Solubility?
- pH Levels — pH measures the amount of hydrogen content in a solution—the more hydrogen ions, the lower the pH and vice versa. Solutions with strong pH levels fully dissociate and those with weak pH levels only partially dissociate. The pKa value is one method used to measure the strength of an acid. A lower pKa value means the drug substance is a stronger acid, which more fully dissociates in water.
- Polarity of Drug and Solvent — Ionization is important for a drug to be soluble for oral drug consumption. In addition, ion trapping is important for the drug to work properly. In the stomach or intestines, the drug is non-ionized so it can be absorbed. When it enters the bloodstream, it needs to become ionized again to prevent it from going back to the GIT and to ensure it is absorbed by the body. Lipid soluble substances contain non-ionized molecules (NaCl), and hydrophilic substances contact ionized molecules (Na+, CL-), meaning the more lipid soluble a drug is, the more absorption there will be. The more water soluble (hydrophilic) a drug is, the less absorption there is.
- Drug Particle Size — The solubility of a drug is directly tied to the particle size. Typically, larger particles are less soluble, especially if the temperature, pressure and polarity for the solutes is the same. The ability for a drug to be soluble allows for simple diffusion of the drug with no energy or carrier protein needed to enter and be absorbed by the bloodstream.
- Solution Process — Most substances are endothermic, or absorb heat in the process of dissolution, meaning an increase in temperature from room-temperature storage to oral consumption and moving into body heat results in an increase in solubility. In addition to temperature, agitation helps increase the speed at which the drug dissolves.
Why is drug solubility important?
- Solubility is one of the crucial properties of a drug and significantly impacts not only assay development, drug bioavailability, absorption and toxicity studies but also drug dosing and drug formulation.
- Researchers in the life sciences therefore need a rapid cost-effective solution to determine solubility before running more costly tests at later stages of the drug development process.
Which component of HPLC is responsible for pumping the mobile phase?
The pump
What type of detector is commonly used in HPLC?
UV-Vis detector
Multiple choice: Which of the following factors can affect retention time? (a) Flow rate (b) Column temperature (c) Mobile phase composition (d) All of the above
d) All of the above
What does the area under the peak in a chromatogram represent?
The concentration of the analyte.
If you change the injection volume from run to run, you need to adjust the calculated concentrations proportionally to maintain accuracy. How do you do that?
Formula for Adjusting Concentration
C_corrected = C_measured x ( V_std/V_actual)
Where:
• C_corrected = Adjusted concentration
• C_measured= Raw concentration from the calibration curve
• V_std = Standard injection volume (from calibration curve)
• V_actual = Actual injection volume used in the sample run
Example Calculation
• Standard injection volume: 10 µL
• Your actual injection volume: 5 µL
• Measured concentration: 50 mg/L
Adjusted concentration:
C_corrected = 50 x (10/5)= 100 mg/L
How do you purge the lines on an HPLC?
Purge: right click, prepare instrument, make sure set to correct lines. If not go to method setup and change to it
- sometimes when run won’t start it’s due to the instrument needing to purge the lines. Hover over the yellow bar over the component (think it’s the binary pump) and it will say it is purging the lines first before starting. If doesn’t work, can manually purge lines as states above
Fill in the blank: The __________ phase is the stationary phase in HPLC.
packing
What is meant by ‘resolution’ in chromatographic analysis?
The ability to separate two peaks in a chromatogram.
Multiple choice: Which of the following is NOT a common type of HPLC column? (a) C18 (b) C8 (c) C2 (d) Phenyl
c) C2
What is the role of the injector in an HPLC system?
To introduce the sample into the mobile phase.
What is a buffer?
A solution that can resist pH change upon the addition of an acidic or basic components. It is able to neutralize small amounts of added acid or base, thus maintaining the pH of the solution relatively stable.
What does the term ‘baseline’ refer to in a chromatogram?
The horizontal line representing the absence of analytes.
Fill in the blank: The __________ is used to maintain a constant flow rate in HPLC.
pump
Which factor primarily influences the choice of mobile phase in HPLC?
The polarity of the analytes.
Difference between FREEBASE POTENCY and SALT FORM POTENCY?
The potency of a drug substance that is a salt form may be reported two ways –
- by its freebase potency, which does
not include the mass of the counter ion,
- or by its salt form potency which does
include the mass of the counter ion.
What does ‘peak width’ indicate in HPLC data?
The efficiency of the separation process.
Fill in the blank: In HPLC, __________ is used to improve separation by varying the mobile phase composition.
gradient elution
What is the importance of column temperature in HPLC?
It affects retention time and peak shape.
- decreasing with increased temperature
- peak shape - improving with higher temperatures
- selectivity - altering the separation of compounds.
Higher temperatures also reduce mobile phase viscosity, allowing for higher flow rates, while also increasing analyte diffusion, both contributing to faster and potentially more efficient separations.
Excessive temperatures can degrade the column, stay within the manufacturer’s recommended range for optimal method development and column longevity.
Why Is Drug Solubility Important?
- Oral dosage is a common drug delivery route thanks to high patient adherence, cost effectiveness, ease of taking the drug, flexibility in design of the dosage and lower sterility constraints.
- A hurdle, however, is designing and formulating a drug with aqueous solubility, drug permeability, dissolution rate, first-pass metabolism, pre-systemic metabolism and susceptibility to efflux mechanisms.
- Poor solubility and low permeability are the most frequent barriers of oral bioavailability of drugs.
- Drug solubility has one of the highest impacts on the desired concentration of a drug in systemic circulation, and therefore whether or not the drug will have the desired impact or pharmacological response in patients. -Drugs that have poor aqueous solubility have a slower drug absorption rate, which can lead to inadequate and variable bioavailability, and render the drug ineffective.
What does ‘signal-to-noise ratio’ refer to in HPLC data analysis?
The measure of the peak signal compared to the background noise.
What is a counterion?
Ions are formed by the addition of or removal of electrons, forming a net positive or negative charge.
A counterion is an ion that is the opposite charge of another ion in solution.
Peptides with free amines are found as positive ions (or cations) and will attract to negative counterions (anions) in solution.
