Hplc instrumentation in detail (Practical) Hplc pump inj_column

HPLC instrumentation in detail

1. HPLC Instrumentation and Techniques in detail 1 Presented by Mr . Pratik P. Shinde M . Pharm II nd Year (Pharmaceutics) University Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University , Nagpur 2021-2022 Guide Prof. Dr . ( Mrs ) Veena S. Belgamwar M. Pharm., Ph.D.

2. HPLC Instrumentation and Techniques Instrumentation Mobile phase reservoirs Construction pump material Pumps Recriprocating Syringe Manual Injection Automated Injection Packings Measure column performance Column care and use Solvent Delivery Sample Introduction Column Packings and hardware Detectors UV-Vis Fluorescence Refractive Index Conductivity Volatametry/Amperometry

3. Solvent supply bottle Chromatographic column Sample Pump Autosampler Detector

4. SOLVENT DELIVERY SYSTEM IN HPLC Mobile Phase Resorvoir. Figure to your right shows a generic version of mobile phase resorvoir. The reservoir that holds the mobile phase is often no more than a glass bottle. Often, the reagent bottle that holds our HPLC solvent can be used as a reservoir. Solvent is delivered from the reservoir to the pump by means of Teflon tubing--called the "inlet line" to the pump Q. What is the purpose of a sinker frit or push filter? --Push filter (10 m m) prevent any dust or particulate matter to enter the pump. --This is because particulate matter can interfere with pumping action, damage valves and seals. In addition, it may also damage the column (by collecting at the top of the column). Bottle capped on outside with a UV- absorbing plastic He degasser Q. Why He degassing of the liquid mobile phase is required? DEGASSING : the practice of removing air from the mobile phase; degassing can be achieved by bubbling He gas into the M.P --He degassing removes dissolve oxygen from the M.P --The presence of oxygen in mobile phase causes bubble formation resulting in air in the flow system and pump pressure will change causing spike in the chromatogram (due to air bubble formation in the detector cell)

5. Requirements for a solvent reservoir are simple: *The reservoir and its attachment to the pump should be made of materials that will not contaminate the mobile phase: Teflon, glass, or stainless steel. *The vessel should have some sort of cap to prevent particulate matter from contaminating the mobile phase. If you are using a solvent bottle as a reservoir, the top of the bottle can be wrapped in aluminum foil to keep dust out or the bottle cap can be drilled to allow inserting the inlet line through the cap. *Don't close the bottle too tightly or removal of mobile phase by the pump will create a vacuum. This prevents mobile phase from flowing the pump, creating a "vapor lock" within the pump. Instead of He degassing vaccum degassing method can also be used

6. B. HPLC PUMP -  The function of a HPLC pump is to pass M.P through the column at a controlled flow rate Pump hardware are shown in Figure to the right Important parts are: Hardware a. Check valves draws the liquid in and out of the solvent chamber Diaphram Coil b. Pulse dampener prevent pulses in the baseline created because of pump strokes

7. (c) Pressure Transducer Monitor the pressure of the liquid flow (d) Flow Controller Controls the liquid flow rate

8. Construction Material of Solvent Delivery System Metallic Non-metallic Steel Titanium PEEK Teflon Ceramic A variety of options are available depending on the needs --Most commonly used pumps are resistant to chemical attack and leaching off the solvent (except HCl) --Low cost, ease of machining and greater operating pressure limit (6000 psi) is an advantage with stainless steel pump                         --Titanium pump has greater degree of inertness Useful for biomolecule analysis ---High cost and tendency to act as ion-exchanger himits its acceptance --PEEK(polyetherethylketone) and Teflon pumps are useful when corrosive solutions such as HCl is used as M.P. Also used for separation of metals

9. Microbore column are used with diameter upto 2 mm (i.d) Narrow column diameter and small size of the packing material requires low flow rate. Reliable separation (<10 m L/min) is difficult unless syringe pump are used. HPLC Pump Classification based on the Flow Rate Microbore Standard bore Preparative (1-250 mL/min) (100mL/min—10 mL/min) (>10 mL/min) - Standard bore is most commonly used for pumping system for both analytical and semi-preparative work (2-12 mm i.d) columns - Preparative column . High flow rate requires special design pump. Upper range for flow rate is usually around 50 mL/min

10. The elution conditions contain the elution profile (isocratic, linear gradient, Column diameter determines to what degree samples are diluted by the system. Smaller diameters result in less dilution and thus higher sensitivity. Specifically, narrow bore columns are 4 to 6 times more sensitive (b) using the injection volume required for a standard bore column (a). © ESA Inc.

11. Pump classification based on the mechanism of eluent displacement Solvent Delivery System Recriprocating piston pump Syringe pump Single Head Multi Head Syringe Pump --Syringe pump provides pulseless solvent delivery (capillary LC) or microbore HPLC hooked to MS. --Larger barrel syringe (10-50 mL) with plunger connected to a digital stepping Motor. As plunger moves forward, it drives the eluent through chromatography with a pulseless flow --- A design of a simple syringe is shown above. What do you think is the major problem with the use of syringe pump? Run time in syringe pump is limited by the volume of the syringe—no flow occurs when the syringe is empty

12. Reciprocating Pumps Why these pumps are called reciprocating? --The piston is driven in and out of the solvent chamber by a gear --Forward Stroke ------  inlet check valve closes and outlet check valve opens and M.P pump to the flowing system ---Backward Stroke-- outlet check valve closes and inlet check valves is refilled Piston Inlet check valve Outlet check valve Single piston pump --Used in HPLC to deliver solutions (e.g., post column derivatization) where pump pulsation is less critical

13. Another approach is to minimize the pulses associated with reciprocating pump. Several approaches can be used Use of in-line pulse dampeners Adjustable spring-loaded dampeners Bellows dampeners Use of two or more pumps, very effective but it is an expensive approach Advantage/Disadvantage of single piston pump --Advantage: cheaper than the dual piston pump. --Disadvantage: rarely used to deliver mobile phase due to the fact that they produce pulse flow

14. Double Piston Pump in HPLC As the name indicates it consists of two pump heads and four check valves --The two piston are driven such that their directions are opposite --Non-circulating gears are continously lubricating the oil chamber which drives the piston so that the forward motion in expelling the solvent takes more time than the reverse motion in drawing the solvent Why do you think the dual piston pump are better than single piston pump? Because dual piston pump minimizes pulsations, it uses two pulgers/two pump heads, which expel solvents alternately First head--  outlet valve opens/inlet valve closes Second head- inlet valve opens/outlet valve closes !st plunger drawing solvent in 2nd plunger drawing solvent out

15. Left and Right chamber delivery While pump pulsation has no effect on separation (resolution),detectability at trace levels is often limited by baseline noise from pump pulsation

16. Your company has decided to manufacture HPLC pump and they are trying to decide what materials to build it with. To a large extent this decision is influenced by the performance criteria that are set by the intended function of the unit. What do you think would be the five most important criteria? Materials should be able to withstand pressure in excess of 7000 psi Inertness---non reactive to sample and solvents 3. Precise flow delivery 4. Reliability---leak free connections and long service life 5. Low raw material cost and machining cost

17. Sample Introduction in HPLC ---The function of injector in HPLC is to introduce the sample in the flowing solvent so that it may be carried to the column What considerations are necessary in injector design? Minimize dispersion or band-broadening (no extra column tubing should be used) b) Allow sample to be introduced without disturbing the solvent flow(otherwise we get baseline disturbances in RI and conductivity detector --Injector may be operated either manually or automatically Manual has two different modes for injecting samples (i) septum mode injection (b) valve injection Septum injection --is the simplest form of injector in the septum device (similar to one used in GC) This allows injecting sample into pressurized solvent stream using a self-elastic septum with a microsyringe Greatest Drawback: limited to max operating pressure of 1500 psi. Therefore, not widely used in HPLC anymore. More useful in low pressure HPLC applications (e.g., protein separations)

18. Valve-Type Injection in HPLC --widely used in HPLC. Allows reproducible introduction of sample into the pressurized M.P without interruption of flow even at high temperature Six-port Valve Type Injector (Used in the laboratory exercise) --One of the simplest and most common valve injector in HPLC is the “six-port valco (or rheodyne model) injector as shown below: Inject valve has two position: (a) Load (b) Inject Valve in the load position . Before injecting the sample the valve is turned to the load position. What is the flow direction in load position? In load position M.P by-passes through the sample Loop and flows straight from the pump through 2 and 3 into the column Why analyte is loaded when the valve is in the load position? This allows to get the sample ready for the next injection. It does not interrupt the flow and will only fill the loop with the new sample. When the valve is switch to inject position the sample is introduced into the flowing stream ----  M.P passess through sample loop from the pump through Port 2 , 1, 4 and 3 into the column 2 3 4 1 5 6 3 1 2 4 5 6

19. What precautions should be taken before the sample is injected? Loop should be flushed with sample using twice the loop volume-- flushing the loop with sample prevents the sample carry over What care you must take during HPLC injection using 6-port valve? --Valve should be rapidly switched from load to inject position. If the rotor is left Halfway flow to the column is stopped and baseline will be disturbed Automated Injection Sample is introduced from a vial held in a sample carousel using a syringe assembly. Valves are automatically Actuated used to wash syringe needle and syringe assembly Benefits of Autoinjector Wide variability in injection Volume (0-1 m L-2 mL) Precision is better than or Equal to manual injection High sample throughput and less labor intensive

20. HPLC Columns Packing Material Container Length Internal Material Diameter Nature Particle of the particle size Container Material --Columns most commonly used at the moment are made with 316 grade stainless Steel (a Cr-Ni-Mo steel, relatively inert to chemical corrosion) Dimensions (Length and Diameter) --Common dimensions are: 4.6 mm (046 cm) internal diameter, 25 cm length, 6.35 mm (0.25 inch) external diameter Range of Length and Diameter Length: 5-30 cm; Internal Diameter: 2-4.6 mm Act as filter prevents particulate to enter the column

21. Fittings from Different Manufactureres --The following figure shows the appearance of different male nuts and ferrules obtained from different manufacturer --The dimension “X” is the distance between the end of the ferrule and the tubing which is different for different manufacturer What are the consequences of having too long or too short of dimension “X” Too Long X: Ferrule does not seat properly and fitting leaks Too Shor X: Get undesirable dead volume in fittings “Rule of Thumb: Tubing and Fittings has to be Matched”

22. High Pressure Plastic Fittings -- A welcome development has been the advent of reliable high pressure plastic fittings and tubings ---Most chromatography suppliers now offer a range of plastic fittings that can be tightened to be leak free, by hand -- Some of the commonly used ones are PEEK (polyethylethyl ketone) as shown below: These fittings are resistance to organic and inorganic liquids except nitric acid, sulfuric acid and THF

23. HPLC Adsorption HPLC: Stationary phase: Unmodified silica HPLC grade silica: microporous particles (3, 5, 7 and 10 micron) Partition HPLC: Normal Phase: Silica is bonded with polar groups chemically Si-(CH 2 ) 3 -O-CH 2 -CH-CH 2 OH OH LiChrosorb Diol Si-(CH 2 ) 3 -CN Bondapak CN Si-(CH 2 ) 3 -NH 2 Polygosil NH 2 Diol > Cyano > Amino

24. HPLC Reverse Phase: Stationary phase: Silica chemically bonded to a low polar functional group Si OH + ClSi R CH 3 CH 3 Si O Si R CH 3 CH 3 R = C 6 H 13 (hexyl), C 8 H 17 (octyl) or C 18 H 37 (octadecyl)

25. Types of Packing Material Used in HPLC Based on the particle size there are three types (a) Pellicular Particles (20-40 m m) (Avg= 30 m m) --have solid inner core with a thin outer surface of S.P --This thin outer surface (1-2 um) is very porous. Hence, aka. superficially porous particle --Used in ion-exchange chromatography --Give high N then porous particles of same size (b) Micro particles (3-10 m m) --Most popular S.P in modern HPLC --Role of pore size is critical. Particles with small pores have high surface area, have greater sample capacity 3-10 m m ( c)Totally porous particles (20-40 m m) --are very porous, more porus than pellicular, may lead to irreversible adsorption. --seldom used as packing material for HPLC due to low N values --Provide ease for dry packing and lower cost (used as guard column and extraction column Currently microparticles are most common in HPLC and are packed with 3-10 m m diameter particles

26. Ethylene bridges Modifying silica particles to include an ethylene bridge gives the particles exceptional chemical stability. (Suitable for UHPLC) High Strength Silica (HSS) and Ethylene Bridged Hybrid (BEH) materials. Particles of both materials, which were designed for the UPLC systems, have diameters in the 1.7–1.8-µm range and feature pores with diameters of either 10 (HSS) or 13 (BEH) nm The hybrid organic-inorganic particles are prepared via copolymerization of TEOS and bis(triethoxysilyl)ethane, are designed to retain silica’s mechanical strength while overcoming pure silica’s tendency to undergo column- damaging (i.e., hydrolysis in alkaline environments , greater than pH 8). BEH’s covalently bonded Si–C–C–Si units render the hybrid material chemically stable up to a pH of 12, which is an ideal condition for analyzing some pharmaceutical agents and other types of compounds. Taken from Chem. Eng News . April-2008, pp 17-23 Packing for Ultra High-Pressure HPLC (U-HPLC)

27. 1-µm particles pack more irregularly in smaller diameter LC capillary columns than in larger (50-µm-diameter column, top; 10 µm, bottom). Yet orderly packing may not yield the best separations. The biggest problem, is the tendency of tiny particles to agglomerate. Use of various solvents and ultrasonication to prepare well-dispersed slurries and then pack capillary columns at high pressure. Packings for UPHPLC

28. Advantage of Using Short Columns with small particle size

29. Effect of Column Internal Diameter on Sensitivity Advantages of using small diameter column --Better sensitivity and higher S/N obtained using a column with small i.d ---Flow rate is slow. This means less solvent consumption, less disposal cost What are the disadvantages of using small i.d columns? --Less loading capacity

30. Nature of the Particles a , k’ Rs Silica based Resin (polymer based) Silica Based Particles D epending on how the silica is treated prior to packing in a column the silanol group (Si—OH gps) present on the silica surface can adapt a number of configurations as shown above Si Si O Siloxane When silica is heated at temp > 800 C, it forms siloxane (devoid of any silanol gps) When silica is heated in temperature range of 200-300 C it allows formation of: Free silanol where the surface silicon atom has three bonds into the bulk structure and the fourth to the OH group Vicinal or associated silanols where two isolated silanol groups attached to two different silicon atoms are bridged by H-bond. Geminal silanols consists of two hydroxyl groups attached to one silicon atom. Geminal silanols are close enough to have H-bond, whereas the free silanol are too far separated. The silanol gps with lower pKa values are believed to be isolated gps with no H-bond with its neighbor

31. Advantages of Silica Packing in HPLC Can withstand high pressure generated when 10-30 cm columns with 3-10 m m particles are used b) Silica is abundant, inexpensive and available in a variety of shapes, sizes and degree of porosity c) Functional group can be readily bonded to silanol and the chemistry of bonding reactions are well understood Limitations of Silica Packing in HPLC --Major limitation is its instability at high and low pH (i.e. above pH 8 or below pH2)

32. Resin or Polymer-Based Packings in HPLC Polymethylmethacrylate based polymers Predominantly used in size exclusion chromatography or ion-exchange HPLC However, resin based reverse phase columns are also commercially available What is the advantage of resin-based packing over silica based packing? ---Stability over a wide pH range (1-13) Disdvantages are: (a) Unstable in presence of organic solvents (b) Polymers swell in the organic solvent

33. Column Care and Use a)The manufacturer’s recommendation regarding M.P pH, flowrates and organic modifier content, temperature as well as maximum operating pressure, etc.; should be followed. General Guidelines b) Use of HPLC grade water and HPLC grade organic solvents, analytical reagent grade chemicals (buffers) must be used to prepare solvents and standards. c) M.P should always be filtered through an appropriate solvent compatible filter (e.g., 0.45 or 0.2 um filter) and vacuum degassed before use. d) M.P flow rate should be altered in small increments (0.2 mL/min-  1mL/min to avoid column back pressure particularly for softer S.P packings e) When not in use store column according to manufacturer’s recommendation Silica based columns

34. Use of Guard Column in HPLC Guard column is used at two points in the HPLC flow system Between pump and the injector -------  Remove particulates and contaminants from mobile phase Pump Guard column Injector (B) Between Injector and analytical column --  Remove contaminants From sample before it enters the column Injector Guard column Analytical column --Guard columns are very short columns (0.5-3.0cm), which are packed with the same S.P as the analytical column except that the particles are bgenerally large to minimize pressure drops Guard columns are used primarily to protect the analytical column from strongly adsorbed sample and matrix. Over time the packing dissolves, resulting in increased backpressure, column voids and broad peaks. Under very harsh mobile phase conditions (above pH 7 and 40°C, and with a buffer salt concen-tration > 50 mM), a silica saturator column should also be used. As the mobile phase passes through this guard column, it dissolves enough silica to saturate (or nearly so) the mobile phase. Then when the mobile phase reaches the analytical column, no additional packing can dissolve. Therefore, column life is extended at high pH. In addition, some HPLC columns are designed for operation at higher pH.

35. Good, economical guard column performance can be obtained with a cartridge type system, as shown in above Figure. The hardware is reused, and only the guard cartridge is replaced. The new, more efficient design minimizes dead volume so that one holder can be used efficiently with analytical (4.6, 3.0 mm id) and narrow-bore (2.1 mm id) columns. The simple design assembles quickly and easily, making it more likely that a guard column will be used and analytical column lifetime extended. When to replace guard columns? A guard column is most effective if it is replaced before the chromatography of the analytical column deteriorates. This can be hard to determine, so many chromatographers select a specific time interval (every week) or sample interval (every 100 samples) for changing the guard column. This is probably the most effective way to minimize contamination of the analytical column. Alternatively, peak shape deterioration and backpressure increases (no more than 10%) can serve as reminders