HPLC Instrumentation Lecture 7 PDF

PHCM561-WS24 Lecture 7 HPLC Instrumentation Prof. Dr. Rasha Hanafi Lecture 7_WS24 1  Competencies By the end of this session, the student should be able to: 2-2-1-1 Compare polarities of different mobile phases. 2-2-1-2 Ident...

PHCM561-WS24 Lecture 7 HPLC Instrumentation Prof. Dr. Rasha Hanafi Lecture 7_WS24 1  Competencies By the end of this session, the student should be able to: 2-2-1-1 Compare polarities of different mobile phases. 2-2-1-2 Identify the cut-off of a mobile phase. 2-2-1-3 Describe sample preparation prior to HPLC. 2-2-4 Use nomograph to find alternative mobile phases. 2-2-3-1 Describe the function of pump, detector, injector. 2-2-3-2 Compare between detectors in HPLC. 2-2-3-3 Describe in detail the set-up of an HPLC system. Lecture 7_WS24 2  Set-Up of HPLC Data acquisition system Sample syringe containing mixture pump HPLC column detector https://www.youtube.com/watch?v=IUwRWn9pEdg Real HPLC in B7- 303 Solvent waste reservoir Response of detector 3 components separated on the Time (min) HPLC column HPLC Lecture 7_WS24 Chromatogram 3  When to use HPLC? 1. Drug discovery: finding new chemical entities (NCE) for adoption as new drug development candidates. 2. Chemical development: developing viable synthetic routes and scale-up processes for synthesizing active pharmaceutical Ingredients (API). 3. Pharmaceutical development: developing dosage forms with optimized delivery and stability profiles for clinical supplies and final products. 4. Drug metabolism/pharmacokinetics (DMPK): evaluating the metabolic and pharmacokinetic profiles of the drug candidates in animal models and human clinical studies. 5. Quality control (QC): assessing the quality of the final manufactured products against published specification for product release. Lecture 7_WS24 4  Set-up of HPLC (real) Filtering and degassing solvents before putting them in the reservoir is a must. Degassers : Filters: Vacuum pumping system to to prevent possible particles in prevent air bubbles in the mobile mobile phase from damaging the phase from reaching the detector pumping or injection systems or causing noise in the baseline of clogging the column. the chromatogram which can make errors in peak area calculation or occlude small peaks. Lecture 7_WS24 5  HPLC vs. classical LC 1. High resolution (i.e. better separation). 2. Small diameter (4.6 mm) stainless steel, glass or titanium columns. 3. Column packing of stationary phase with very small (3, 5 and 10 μm) particles. 4. Relatively high inlet pressures and controlled flow of the mobile phase. 5. Continuous flow detectors capable of handling small flow rates and detecting very small amounts. 6. Rapid analysis. HPLC vs. UPLC? Lecture 7_WS24 6 What are the components of an HPLC 1. Mobile phase reservoirs 2. Pumps 3. Injection port 4. Column 5. Detector Lecture 7_WS24 7  SOLVENTS FOR HPLC MOBILE PHASES It ranks solvents by their relative abilities to displace solutes from a given adsorbent Below this  the solvent exhibits absorbance. So the “cut off” is the shortest  where a solvent can be used without interfering with UV detection. Lecture 7_WS24 8  MOBILE PHASES IN HPLC  Single solvents Choice: according to eluotropic series. Purity: HPLC grade (even better than analytical grade!) Degassing; mandatory before use. Water: purification immediately prior to use ("HPLC water").  Mixtures note! "40% B" refers to the volume of B before the solvents are mixed binary (or higher) mixtures of organic solvents, water and buffers in various combinations “NOMOGRAP H" for comparison of eluent strength, the vertical line indicating Lecture 7_WS24 9 same 0  PUMPS Should produce steady and reproducible flow. A fluctuating flow creates detector noise that obscures weak signals. Requirements of pumping system: 1. Pulse free output. 2. Flow rates from 0.1-10 mL/min. 3. Flow control and flow reproducibility of a Gradient construction: maximum error of 0.5%. 1. High-pressure gradient 4. Corrosion resistant 2. Low-pressure gradient (more common) (stainless steel & Teflon). where pro-portioning of the solvents (up 5. Generation of pressure up to 4) through a four-way valve at low to 6000 psi (400 Bar). pressure takes place, then pumping the mixture at high pressure into the column is performed. Lecture 7_WS24 10 RECIPROCATING PUMP to column to pulse damper ball check valves solvent from reserv oir Lecture 7_WS24 11  The Column  Analytical column: length: 5 – 30 cm. ID: 1 – 5 mm. steel or plastic. expensive (>8000 L.E.) easily damaged by dust or particles in the sample or solvent. Protection: 1. by periodically Preparative column  : renewed guard industrial scale (for 1 kg of columns material, volume 300 L) containing the same stat. phase like main column. Lecture 7_WS24 2. by passing12 solvents and  INJECTION PORT Most common injection valve: six-port or Rheodyne™ Syringe: valve blunt needle to avoid damage to injection port When the valve is The syringe is used rotated 60° to wash and load counterclockwise, the the loop with fresh content of the sample sample at loop is injected into atmospheric the column at high pressure. pressure. Lecture 7_WS24 13  SAMPLE PREPARATION BEFORE INJECTION In general, the “dilute and shoot” approach can be used for most drug substances and parenteral products. A common process of “grind → extract → dilute → filter” is used for most solid dosage forms such as tablets or capsules. More complex dosage forms, such as The sample is put in an suppositories, lotions, and creams, HPLC vial containing the and physiological samples (serum or final testing solution with plasma) might require additional the extracted analytes sample clean-up and extraction such ready for HPLC analysis. as liquid-liquid extraction or solid- Lecture 7_WS24 14 phase extraction (SPE).  DETECTION REQUIREMENTS OF A DETECTOR: 1. Sensitive to low concentrations of every analyte. 2. Small volume to avoid peak broadening. 3. Linear response. 4. Insensitive to changes in temperature or solvent composition. A 1982 survey of 365 published papers revealed that 71% were base upon detection by UV Lecture absorption. 7_WS24 15  UV AND FLUORESCENCE DETECTORS UV detector: Fluorescence THE FLOW CELL detector: A special type of UV Fluorescence is detectors is the measured after photodiode array excitation of the eluate detectors (PDA) with a laser. records the Adv.: very sensitive. absorbance Disadv.: response of Derivatization to spectrum of each few analytes. originally non-fluorescing solute as it is analytes by covalent eluted, besides attachment of fluorophors showing its peak in to analytes either The linearity the a. prior to range is 5 orders chromatogram. chromatographic of magnitude. separation b. adding derivatization The flow cell is typically Z-shaped. Inreagents order totominimize the eluate the between extra column broadening, the volume is kept as small as column and possible (1-10 μL) and cellLecture lengths to 2 detector. 7_WS24 to 10 mm. 16  REFRACTIVE INDEX DETECTOR  Useless in gradient elution, sensitive  Responds to all to T and P changes.  Small linear range only analytes, why?  Detection limit 1000x poorer than that of UV detector Lecture 7_WS24 17  References 1. “Principles of instrumental analysis, 5th ed. by Skoog, Holler, Nieman” Chapter 29. 2. “Quantitative Chemical Analysis, 7th ed. By Harris” Chapter 25. 3. “Modern HPLC for Practicing scientists” by Michael W. Dong, © 2006 by John Wiley & Sons, Inc., New Jersey. 4. http://www.studyhplc.com 5. http://hplc.chem.shu.edu/HPLC/index.html 6. Lecture of “Chromatography-II” by Dr. Raimund Niess, GUC, 2009. Lecture 7_WS24 18