Hplc Interview Questions 6z3i6

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1. Question 1. What Is Hplc? Answer : HPLC stands for High-Performance Liquid Chromatography (formerly referred to as High-Pressure Liquid Chromatography). It is a chromatographic technique used to separate the components in a mixture, to identify each component, and to quantify each component. In general, the method involves a liquid sample being ed over a solid adsorbent material packed into a column using a flow of liquid solvent. Each analyte in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analytes. If the interaction is weak, the analytes flow off the column in a short amount of time, and if the interaction is strong, then the elution time is long. 2. Question 2. What To Do When Back Pressure Increases? Answer : o An increase in back-pressure usually suggests either a guard or analytical column problem. To find exactly where the problem lies we suggest you remove the guard column (if you are using one) and replace the old cartridge with a new one. o If the original pressure is restored, you solved the problem. o If the pressure remains high, disconnect the analytical column from the system, backflush it (do NOT connect the column to the detector while doing so) and run a few column volumes of your mobile phase through the column. o If the problem still persists you may have some strongly retained contaminants in your column coming from your previous injections. o Run the appropriate restoration procedures, as suggested by the column manufacturer, and retest the column. o If the initial pressure is not restored you may have to change the inlet frit or replace the column. o Always run your system (2 to 5 ml/min) without the guard column and the analytical column to that your pressure isn’t coming from another source, like a blocked in-line column prefilter, blocked/kinked tubing, particulates blocking your injector etc. o Always work your way from the detector back to the pump to isolate the problem. 3. Question 3. How Do I Determine The Void Volume In Hplc? Answer : The void volume of a system is usually determined by injecting an unretained standard (Uracil in RP-HPLC) that has no or very little retention on a particular phase. Slight variations in this value are explained by the extra column dead volume of your specific system configuration and set-up. Multiply the elution time of the unretained compound by the flow rate to get the actual void volume of the system and column. To determine the column void volume alone you would need to subtract the system void volume determined without the column attached.

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4. Question 4. Why Should I Use A Guard Column With My Analytical Or Preparative Column? Answer : o A guard column is recommended to protect the analytical/preparative column from contamination from particulates from the injection, debris from worn pump seals/injector rotor seals or unfiltered mobile phases. o Filtration through a 0.22um to 0.45 um should be done in order to remove particles and help degas the mobile phase at the same time. o Solid Phase Extraction or Liquid Liquid Extraction also help produce a cleaner sample for direct injection. o Failure to use a guard column directly exposes the analytical or preparative column to contamination and therefore reduces its practical lifetime. o Care should be taken to use, whenever possible, the same material in the guard column as in the analytical/preparative column especially when doing method development. o Typical analytical guard columns are 1 or 2 cm long with either 2.0 (2.1) or 4.0 (4.6) mm depending on the column dimensions and 1cm long for 10mm, 21.2mm & 30mm column id. o Whether you opt for 1 or 2 cm long guard columns is tied to how harsh your mobile phase is and how messy your sample is. Ideally, in order to keep your chromatography and to avoid increasing the system pressure, you should use the shortest guard column available and use the same id as the column whenever available. Otherwise you should choose the closest smaller id guard column available. 5. Question 5. What Happens If My Sample Solvent Is Stronger Than My Mobile Phase? Answer : We do not recommend injecting in a stronger solvent because it usually results in peak distortion, broadening, poor sensitivity, and shortening of retention times. This happens because some analytes will tend to travel too quickly through the column, instead of eluting in a symmetrical band. If you absolutely must do this, keep the volume as small as possible and make sure the solvents are miscible. 6. Question 6. Can I Get A Sharper Peak By Injecting My Sample In A Weaker Injection Solvent? Answer : o In this scenario, the sample is initially concentrated onto the head of the column and moves through the column in a tight band. o This technique is sometimes used to minimize band broadening for a larger volume sample injection. o Keep in mind that your sample components must be soluble in the mobile phase as well, in order for this to work. 7. Question 7. How Much Sample Can I Inject On My Lc Column? Answer : Two different types are possible: o Mass overload (too much analyte injected on the column) o Volume overload (too much liquid injected on the column) The chromatograms are somewhat different in these 2 situations.

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In mass overload, the analyte molecules saturate the silica at the inlet end of the column which causes the excess molecules to move forward down the column without much interaction, reducing in turn the analyte retention time and showing a "shark fin" peak shape (fronting). Volume overload occurs when the injected sample volume is large enough to carry analyte molecules through a significant proportion of the interstitial volume within the column and leads to shark fin type peaks as well and later elution times. 8. Question 8. What Size Threads Are On The End Fittings Of My Hplc Column? Answer : Most fittings on your HPLC and UHPLC systems and columns have 10-32 threads. However, you will find that fittings and columns from Waters, Rheodyne and SSI (Lab Alliance) have different seating depths. 9. Question 9. What Is The Internal Diameter Of My Lc Tubing? Answer : The internal diameter of HPLC grade stainless steel tubing is identified by the color coded band on the pre cut tubing while HPLC PEEK tubing is also colored according to its internal diameter. Typical encountered colors used for 1.16” od HPLC tubing (color coded band for SS or solid color for PEEK) are: o Black = 0.004” ID o Red = 0.005” ID o Yellow = 0.007” ID o Blue/Tan = 0.010” ID o Orange = 0.020” ID o Green = 0.030” ID Please note that these colors may differ depending on the manufacturer especially when it comes to HPLC stainless steel tubing. Please check with your tubing supplier/manufacturer to confirm tubing ID color coding. Please note that the HPLC stainless steel tubing comes in precut lengths as it is virtually impossible to produce smooth, clean, bur-free cuts without the manufacturer’s precise machinery tools. As for SS in HPLC, it also becomes extremely difficult to produce, in-house, bur-free, perfect cuts especially when using the narrower IDs (less than 0.005” ID). 10. Question 10. How Much Should I Change My Injection Volume If I Change The Size Of My Column? Answer : Optimal injection volumes are directly related to the cylinder volume of your column and are, therefore, dependent on the cross sectional area (A=π r2) and length (L) of your column. Since that is the case, you can estimate any adjustment from an existing method for injection volume. If you are converting to a different size ID (with packing material and length remaining the same), just multiply your current volume by the ratio of the radii squared to determine the correct volume for your new method. For example, if you are currently injecting 20 µL on a 150 x 4.6 mm column and then switch to a 150 x 3.0 mm column, you could estimate the adjusted volume by multiplying 20 x (1.52)/(2.32). Your new volume should be about 8.5 µL. 11. Question 11. Can I Get A Sharper Peak By Injecting My Sample In A Weaker Injection Solvent (such As 100% Water For Reverse Phase)? Answer :

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In this scenario, the sample is initially concentrated onto the head of the column and moves through the column in a tight band. This technique, referred to as “on-column compression” or “point-injected”, is sometimes used to minimize band broadening for a larger volume sample injection. Keep in mind that your sample components must be soluble in mobile phase as well, in order for this to work. 12. Question 12. How Do I Address An Increase In Backpressure And Other Signs Of Blockage Or Contamination? Answer : Please follow the recommendations in our blog “Building up Pressure on HPLC?”. This will help you identify or confirm the source of pressure. If the column itself is found to be the source of difficulty, please follow the column regeneration steps given on our LC Column Cleaning Recommendations page. To help avoid future occurrences, Restek highly recommends the use of guard columns to protect your reversed phase and normal phase analytical LC columns from both frit blockage and column contamination. 13. Question 13. How Do I Determine Total Column Volume Or Void Volume For Lc? Answer : The term “column volume” usually refers to the void volume, which represents the volume of mobile phase that is between the silica particles. This area is referred to as the interstitial space. You can estimate void volume by multiplying the total column volume (pi x radius2 x length) by a factor that estimates the typical packing efficiency for a particular column type. For fully porous columns, the equation for void volume (in mL) is V = (0.68) pi r2 L, where V = column volume in mL, r = column radius in cm, and L = column length in cm. For superficially porous columns, such as our Raptor columns, the factor is different and the equation is V = (0.50) pi r2 L. Void volume is more commonly estimated experimentally by injecting a standard containing an analyte that is known to have no, or negligible, retention on that particular column phase. A good example of this for reversed-phase HPLC is uracil. One should be aware that this estimation is also subject to extra column dead volume for the specific instrument that is being used, so it may vary slightly. 14. Question 14. Why Am I Seeing Bleed From My Biphenyl Column On My Uv But Not On My Mass Spec? Answer : A small amount of phase bleed is inherent for all phases, including phenyl phases, and is somewhat dependent on the size and dimensions of the column. This bleed is usually negligible and does not affect retention times, but may be visible, particularly by UV detection. It can often be reduced after conditioning. Bleed may also be minimized by using an isocratic elution, a shallower gradient, and/or incorporating a gradient flush between runs. 15. Question 15. What Should I Use To Analyze Explosives (as Per Epa Method 8330b) By Hplc? Answer : While no one LC column can provide baseline separation for all of these analytes combined, the Raptor Biphenyl and Raptor ARC-18 columns from Restek are an outstanding choice for primary and confirmation analysis. Fully porous HPLC particles, namely the Ultra C8 and Ultra Aromax columns, are also an option. Keep in mind that a variety of column phases may provide partial solutions for this method, but Restek has found these pairs to give optimal results.

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16. Question 16. Is Special Conditioning Needed For The Raptor Biphenyl Column Prior To Its First Use, Or If It Has Been Sitting Idle? Answer : For the most part, the Raptor Biphenyl column behaves just like any other reversedphase column. However, in certain circumstances, longer equilibration times may be needed. Switching between organic solvents, such as acetonitrile and methanol, may require a 15-20 minute flush in high organic mobile phase. 17. Question 17. How Much Equilibration Time Is Required In Between Gradient Runs On A Raptor Biphenyl Column? Answer : Whether you are using fully porous silica or SPP silica, some equilibration time is needed between runs if you are using a gradient and the amount of time is similar for both types of columns. Usually, the equivalent of 7 column (void) volumes is sufficient unless you are using an ion-pairing technique. 18. Question 18. What Mobile Phase Solvents Are Compatible With Spp Or Raptor Columns? Answer : Any solvent that is commonly used for reversed-phase LC will work fine, including but not limited to water, methanol, and acetonitrile. 19. Question 19. Can I Pump Solvent Through The Raptor Biphenyl Column Backwards To Clean It? Answer : Similar to UHPLC columns, it is not recommended to reverse the flow for these columns. However, you can still pump through a series of solvents, as long as they are miscible. 20. Question 20. How Much Can I Inject Onto A Raptor Column? Answer : Injection volume depends on a number of factors including column dimensions, sample solvents, and analysis requirements. As is always a good practice with chromatography, try to inject as little as possible and in the same or weaker solvent than your mobile phase. 21. Question 21. How Do Raptor Arc-18 Columns Differ From Ordinary C18s? Answer : The significant difference is the ruggedness of the bonded phase. With the ARC-18, any residual silanol groups are shielded and made inert through steric protection. The result is a wider operating pH range of 1.0–8.0. The ARC-18 is particularly useful between a pH of 1.0 and 3.0, where other C18 column phases may begin to degrade under these harsh conditions. Like the Raptor Biphenyl column, the stationary phase is bonded to superficially porous silica particles (SPP). 22. Question 22. How Well Does The Raptor Arc-18 Column Work For Acids And Bases? Answer : The ARC-18 provides added retention for charged bases and, in many cases, is preferred over a conventional end-capped C18. For neutral acids, it works well and is preferred over end-capped C18 phases, particularly at pH < 3. The ARC-18 also works for neutral bases and charged acids, but provides more advantages and performs best at the lower pH ranges.

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23. Question 23. Can The Raptor Arc-18 Column Be Used With 100% Aqueous Mobile Phases? Answer : No. We recommend using the Raptor ARC-18 column with at least 5% organic in the mobile phase. For applications requiring higher aqueous content, we suggest the Ultra Aqueous C18 or Pinnacle DB Aqueous C18 columns. 24. Question 24. How Do We Know That The Raptor Columns Are Rugged? Answer : We use frits that are less prone to clogging from sample matrices, and the column packing is less likely to be damaged by higher pressures that might develop. Added protection of a guard column is also available and recommended to extend the life of the column further. 25. Question 25. Which Fittings Can Be Used For Uhplc? Answer : You can either use the stainless steel fittings that are like the ones that come with your UHPLC system or you can use EXP fittings. The EXP fittings can be used up to 20,000 psi when tightened with a wrench. In any case, always make sure you are using a fitting with zero dead volume so that the high efficiency provided by UHPLC is not compromised by extra dead volume. 26. Question 26. How Do I Tighten My Fittings? Answer : Our polymer-based universal connectors and PEEK union connectors only need to be hand-tightened, whereas any of the stainless steel fittings need to be wrenchtighten. The EXP fittings can be used either way: They are hand-tightened for use up to 8,700 psi or wrench-tightened for use up to 20,000 psi. Note that over-tightening causes galling and will destroy the threads. Fittings that need to be wrench-tightened generally require ¼-turn past hand tight to achieve a leak-tight seal. Unfortunately, there is no universal torque setting. 27. Question 27. What Injection Solvent Should I Use For Hilic Separations? Answer : The injection solvent should be as close of a match as possible to the initial mobile phase conditions, which are high in organic content for HILIC separations. By matching the injection solvent to the initial mobile phase conditions, you get better peak shape, increased retention, and higher sensitivity. 28. Question 28. What Kind Of Ph Effects Do I Have To Be Aware Of With Hilic Separations? Answer : The effect of pH on analyte charge state varies based on each compound’s pKa, to pH effects must be evaluated carefully during method development. With HILIC methods, the high concentration of organic solvent in the mobile phase raises the pH, and the actual eluent pH can be 1–1.5 units higher than in the aqueous portion alone. The charge state of the column itself can also be affected. For example: in a Raptor HILIC-Si column, the bare silica has a pKa between 3.8 and 4.5, so the mobile phase pH changes the charge of the silica surface, making it neutral in very acidic conditions and ionized (negatively charged) as the pH begins to approach 3.8 and above. For this reason, if your analyte has one or more protonated amine or quaternary amine groups, it’s a good candidate for analysis on a Raptor HILIC-Si column.

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29. Question 29. Can I Use Buffers For Hilic Separations? What Kind And What Concentration? Answer : Many HILIC separations use a mass spectrometer for the detector, so volatile buffers like ammonium formate and ammonium acetate are very common. However, the high organic content of the mobile phase can cause buffer salts to precipitate, which can lead to downtime for instrument maintenance. In addition, high buffer concentrations can impact chromatography by reducing analyte retention. To avoid these effects, method optimization is required and 10 mM is a good starting point for buffer concentration. Both the A and B mobile phases should be buffered equally in order to keep the ionic strength constant during a gradient for the most consistent MS detector response. Check with your MS vendor for the maximum buffer concentration they recommend for your ESI source.

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1. Question 1. What Are The Main Differences Between High Performance Liquid Chromatography And Gas Chromatography? Answer : o In HPLC the mobile phase is a liquid whereas in Gas Chromatography the mobile phase or carrier is a gas. o HPLC is useful for analysis of samples which are liable to decompose at higher temperatures. GC involves high temperatures so compounds are stable at such temperatures. o Gas Chromatography is applied for analysis of volatile compounds whereas non volatile compounds can be easily analyzed on HPLC o Gas Chromatography cannot be used for analysis of high molecular weight molecules whereas HPLC has applications for separation and identification of very high molecular weight compounds o HPLC requires higher operating pressures than GC because liquids require higher pressures than gases for transport through the system o HPLC columns are short and wide in comparison to GC columns 2. Question 2. Which Type Of Gc Detector Is Most Commonly Used? Explain Its Working Principle And What Are Its Limitations? Answer : The most commonly used detector is the flame ionize detector. The sample is combusted with the help of fuel gas and oxidant in the detector body. Combustible sample components burn and produce ions and electrons which can conduct electricity through the flame. A large potential difference is applied at the burner tip and the collector electrode located above the flame and the current between the electrodes is measured. The detector is mass sensitive and response is not affected by carrier gas flow rate changes. However, the detector is not responsive to inorganic gases such as CO, O2, NH3, N2, CS2, CO2, etc. 3. Question 3. What Are The Commonly Used Carrier Gases In Gc Analysis When Using Fid Detector? Answer : Inert gases commonly used in analysis when using FID detector are Nitrogen and Helium. Nitrogen is more commonly used as it is less expensive than Helium. Purity of carrier gas should be more than 99.995% and on-line traps should be used to prevent residual moisture or other impurities from entering the system. 4. Question 4. What Are The Desirable Characteristics Of A Gc Detector ? Answer : The detector chosen for particular analysis should : o Give reproducible response to changes in concentration of eluting compounds in the carrier gas stream. o Should provide a large linear dynamic range o Should have high sensitivity o Should have small internal volume to give narrow peaks and also facilitate flushing of previous sample traces o Should preferably be non-destructive

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5. Question 5. What Do You Understand By Specificity Of A Detector? Answer : Detectors falls into three categories depending upon response to the eluting compounds: o Non-selective – Respond to all component in the gas stream except for the carrier gas o Selective – Respond to a particular class of compounds with common physical or chemical properties o Specific – Respond to a single specific compound only in the carrier gas stream 6. Question 6. What Are The Commonly Used Types Of Capillary Columns? Answer : Capillary columns are generally 10 – 100m long tubes having an internal diameter ranging from 0.1 – 0.5mm made of flexible material such as fused silica. Common types of capillary columns are o Wall coated open tubular (WCOT) – Internal wall is coated with a very fine film of adsorbing liquid o Surface coated open tubular (SCOT) – Inner wall is lined with a layer of solid on to which the liquid phase is absorbed. The columns are flexible and wound into several turn coils ed on a SS cage inside the column oven 7. Question 7. What Do You Understand By Column Efficiency And How It Is Expressed? Answer : On continuous use a column gradually loses its original resolution power. Column efficiency is expressed on the basis of plate theory concept. Each component under separation spends a finite time in each theoretical plate. Smaller the plate height the larger the number of plates (N) and better is the column efficiency. 8. Question 8. What Do You Understand By Temperature Programming In Gc Analysis? Answer : Temperature programming means change of temperature of the column at a rate predetermined rate during the analytical run. This has the same influence on elution time of separated components as gradient programming in HPLC analysis. Temperature programming helps reduce analysis time by permitting early elution of less volatile components. 9. Question 9. When Is Isothermal Operation Useful? Answer : Isothermal operation is useful when high resolution is required for separating compounds having narrow boiling range. Temperature is set to around mid range of boiling points of constituents. This results in good resolution of low boiling components but band broadening of higher boiling components can result due to their longer retention in the column.

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10. Question 10. What Measures You Would Adopt To Extend Useful Life Of A Column? Answer : o Condition a column before first use or after long time storage o Take care not to exceed upper temperature limit specified by the manufacturer o Avoid injection of solutions which are strongly acidic or basic in nature o Rinse columns by injection with blank solvents such as methanol, methylene chloride or hexane to remove contamination of column after excessive usage 11. Question 11. What Is The Basic Principle Of Paper Chromatography? Answer : Paper chromatography is a form of liquid chromatography where the components of a mixture of organic compounds get separated as unique spots by unidirectional flow of the developing liquid mobile phase solvent mixture over the filter paper to which a spot of the sample is applied. The distance travelled by each component is specific under the given set of operational conditions. 12. Question 12. Why The Developing Solvent Mixture Is Prepared Fresh Before Use? Answer : The developing liquid phase comprises of a pure solvent but more often it is a mixture of two or more solvents in specified proportions. In case solvents are mixed and stored for long periods there could be loss of volatile component which will alter the mixing proportions. 13. Question 13. Why Is It Necessary To Cover The Developing Chamber During The Paper Development? Answer : During the chromatogram development chamber is covered.This is essential as the environment inside the chamber should remain saturated with the solvent vapour. Development times can vary from about an hour to several hours and a saturated environment prevents losses due to evaporation. 14. Question 14. What Are The Common Techniques Used For Detecting Colourless Spots? Answer : It is easy to distinguish coloured spots visually but for colourless compounds alternate techniques need to be adopted which can be specific or non-specific. o A common non-specific method is suspension of developed chromatogram in iodine vapour. Most organic compounds show up as brown spots. o The sheet is viewed in a UV Viewing cabinet under 366 nm and 254 nm wavelength lamp illumination. On observation the spots need to be carefully marked with a pencil for Rf calculations. o Under specific methods amines and amino acids are observed by spraying heated paper on development with 0.2% hydrazine. Deep blue or purple spots begin to appear. o Alkaloids – Dragendroff’s reagent spray results in orange or orange yellow spots. o Aldehydes&Ketones – 2,4-DNPH spray in methanol and sulphuric acid results in orange or yellow spots.

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15. Question 15. Why Should The Samples Have Reasonable Solubility Which Is Neither Too High Or Too Low In The Developing Solvent Mixture? Answer : The samples should have a medium solubility in the developing solvent mixture.Too high a solubility will lead to transfer of the component alongwith the solvent front and on the other hand if the solubility is too low the component will not be carried by the solvent mixture and will remain close to the initial applied spot. In either case the resolution of the mixture components will be low. Thus reasonably good resolution can be obtained for medium solubility of compounds in the solvent mixture. 16. Question 16. What Information You Get From The Retardation Factor Value? Answer : Retardation factor Rf is a measure of the separation of a particular component. It is expressed as Rf = distance moved by the component spot/ distance moved by solvent front Rf is a unit less quantity and lies between 0and 1.A value of 0 indicates no separation has taken place and 1 represents that the component has moved entire length alongwith the solvent front. In case two spots have same value of Rf it indicates that they are not resolved. At least a difference of 0.05 is necessary to discern the separation between two spots. 17. Question 17. Can You The Various Paper Chromatography Techniques? Answer : Paper chromatography separations are classified in accordance with the direction of flow of mobile phase along the filter paper. o Ascending paper chromatography – the carrier liquid moves from bottom upwards. o Descending paper chromatography – the carrier liquid trough is on top and mobile phase moves downward on the filter paper. o Ascending – descending paper chromatography – The paper is rolled downward over the rod at the top. On reaching the top in ascending mode it starts downward movement in the next phase. 18. Question 18. What Are Essential Criteria For Selection Of Suitable Solvents For Paper Chromatography? Answer : Solvents are selected on the basis of solubility of the sample components. In general it is advisable to keep in mind: o Solvents are not toxic or carcinogenic. o Solvent constituents of mixture should not react with any of the sample constituents. o Solvents selected should not interfere in detection of separated spots. o Solvents should not be highly volatile as loss of components can result in change of mixture composition.

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19. Question 19. Why Paper Chromatography Has Retained Its Applicability In The Face Of A Emergence Of Advanced Instrumental Techniques? Answer : Chromatographic technique of analysis has seen an impressive growth over time. Such advances have increased laboratory throughputs lowered limits of detection and has made forays into new areas of applications. Paper chromatography has retained its ground till date and is popular in laboratories across the world. Some of the reasons for this are: o Low cost of analysis and freedom from maintenance. o Separated spots are visible for coloured compounds and colourless compounds can be viewed by using alternate techniques. o Minimum operation and training requirements.Solvent consumption is much less as compared to more sophisticated techniques. o Paper chromatography serves as a good demonstration of basic concepts of separation for school and undergraduate students. 20. Question 20. What Are The Limitations Of Paper Chromatography Technique? Answer : Paper chromatography has some limitations such as: o Semi-quantitative in nature. o Overlapping of spots of components having close Rf values. o Higher concentration of components often leads to streaking instead of welldefined spots. o Errors in Rf calculations can result from uneven flow of solvent front. This can be caused by running out of solvent at the bottom of the chamber, uneven cutting of the filter paper or unevenness of the bottom of the development chamber. o Improper sample spotting, spotting below the marked line resulting in dipping into the solvent or accidental dipping of spot into solvent while inserting the paper into the solvent chamber. 21. Question 21. Which Type Of High Performance Liquid Chromatography Technique Is Most Widely Used? Answer : Reverse phase Chromatography has the widest range of applications. The stationary phase comprises non polar organic chains bound to inert silica surface and mobile phase comprises of aqueous or aqueous-organic mixtures comprising of polar solvents of varying degrees of polarity. The elution sequence is polar followed by less polar and least polar or non polar compounds eluting last through the column. 22. Question 22. What Is The Separation Principle In Size Exclusion Chromatography? Answer : In size exclusion chromatography the separation does not involve chemical interactions between eluting molecules and stationary phase. The separation takes place on the basis of molecular size with larger molecules eluting first and small molecules in the end. Small molecules are retained longer in the pores of the stationary phase therefore they get eluted last.

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23. Question 23. Why Is It Necessary To Degass The Mobile Phase? Answer : Mobile phases entrap air from the atmosphere and this trapped air gets released as small bubbles under high pressures encountered during the HPLC analysis. Such bubbles can lead to noise in detector response or hinder flow of mobile phase through columns. In order to overcome such problems degassing of mobile phase becomes essential. 24. Question 24. Which Is The Most Commonly Used Detector In High Performance Liquid Chromatography And Why? Answer : The most commonly used detector in HPLC is the UV-VIS detector. The reason for its predominant use is that it gives specific response to a particular compound or class of compounds. Most of the organic compounds absorb at specific wavelengths covered in the available wavelength range of the detector. 25. Question 25. What Do You Understand By A Bulk Property Detector And A Specific Property Detector? Answer : A bulk property detector responds to some property of mobile phase and sample combination ing through it at any point of time such a Refractive index or Electrochemical detector whereas a specific property detector is responsive only to the characteristic property of the eluting molecule and is independent of changes in mobile phase composition such as UV-Vis and Fluorescence detectors. 26. Question 26. What Do You Understand By Isocratic And Gradient Elution? Answer : When the composition of the mobile phase is not changed through the chromatographic run the operation is termed as isocratic. It can involve a single solvent or a mixture of two or more solvents mixed in a fixed proportion. In gradient operation the composition at start of run is programmed to change at a predetermined rate and the composition at the end of run is different from the composition at the start. 27. Question 27. What Are The Desirable Features Of A High Performance Liquid Chromatography Detector? Answer : The desirable features of a detector are o Sensitivity towards solute over mobile phase. o Low dead volume to eliminate memory effects o Low noise o Low detection limits o Large dynamic linear range 28. Question 28. What Do You Understand By Theoretical Plate Concept And How Hetp Affects The Separation Of Hplc Column? Answer : Plate theory concept was introduced to explain efficiency of columns. The concept assumes that a state of instantaneous equilibrium exists between the concentration of solute in stationary phase and the mobile phase and further the column is imagined to be divided into a number of theoretical plates. Any analyte spends a finite time in each plate and this is the equilibrium time. Smaller the plate height the greater is the number of plates in a given length (HETP) and better is the column resolution.

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29. Question 29. What Are The Benefits Of Fast Lc Or Uhplc? Answer : Fast or UHPLC technique makes use of small particles below 2 μ size Use of such particle sizes result in high resolution and as small columns can be used it results in completion of analysis in much less time thereby reducing consumption of expensive solvents. We hope you had a great learning experience through the introductory free e-learning HPLC course. I shall remain in with you for our offerings on advance versions of the HPLC e-learning courses and subsequent introduction covering other analytical techniques. 30. Question 30. What Is Chromatography? Answer : It is technique for rapid and efficient separation of components of a mixture and purification of compounds. It is based on differential migration of the various components of a mixture through a stationary phase under the influence of a moving phase. 31. Question 31. What Is The Basis (principle) Of Chromatographic Process? Answer : It is based on the differential migration of the individual components of a mixture through a — stationary phase under the influence of a moving phase. 32. Question 32. What Type Of Solvents Are Generally Employed In Chromatography? Answer : Generally solvents having low viscosities are employed in chromatography. This is due to the fact that the rate of flow of a solvent varies inversely as its viscosity. 33. Question 33. Name Some Chromatographic Techniques? Answer : Paper chromatography, column chromatography, thin layer chromatography, gas chromatography. 34. Question 34. What Are The Moving And Stationary Phases In Paper Chromatography? Answer : Water absorbed on cellulose constituting the paper serves as the stationary phase and organic solvent as moving phase. 35. Question 35. What Is Meant By The Term Developing In Chromatography? Answer : During chromatography, if the components to be separated are colourless, then these separated components on chromatogram are not visible. Their presence is detected by development, which involves spraying a suitable reagent (called developing reagent) on the chromatogram, or placing the chromatogram in iodine chamber when various components become visible. This process is called developing of chromatogram. 36. Question 36. How Does The Liquid Rise Through The Filter Paper? Answer : By means of capillary action.

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37. Question 37. What Is Meant By The Term Rf Value? Answer : Rf (retention factor) of a substance is defined as the ratio of the distance moved up by the solute from the point of its application to the distance moved up by the solvent from the same point. 38. Question 38. On What Factors Does The R Value Of A Compound Depend? Answer : o Nature of the compound. o Nature of the solvent. o Temperature. 39. Question 39. Give The Biochemical Uses Of Chromatography? Answer : It helps in the separation of amino acids, proteins, peptides, nucleic acids, etc. 40. Question 40. Name The Scientist Who Introduced Chromatographic Technique? Answer : Russian botanist M. Tswett (1906). 41. Question 41. What Are The Advantages Of Chromatography Over Other Techniques? Answer : o It can be used for a mixture containing any number of components. o Very small quantities of the substances can be effectively detected and separated from a mixture. 42. Question 42. What- Is Loading (or Spotting)? Answer : The application of the mixture as a spot on the original line on the filter paper strip or addition of mixture to the column, is called loading (or spotting). 43. Question 43. What Are The Essential Characteristics Of The Substance Used As A Developer? Answer : o It should be volatile. o It should impart colour to the different spots. o It should not react with various compounds which are being separated.

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