HPLC and FPLC: Troubleshooting and Standardizing Chromatogram Purification Profiles

Table of contents

1. HPLC and FPLC

2. Beer Lambert law

3. Theoretical plates

4. Calculationoftheoreticalplates

5. Resolution

6. Phases

7. Calculation of retention time and absorbance

8. Column capacity

9. Components

10. Pumps

11. Flow path

12. Tubings

13. Leak

14.System wash

15.1njector

16. UVdetector

17. Gaurd column wash

18. Column

19.Standards for column

20. Normal phase column

21. Reversephasecolumn

22. Column wash

23.Solvent

24. Buffer selection for reverse phase HPLC

25. Preliminary purificatino

26.Sample preparation

27. HPLC sample

28. Gradient

29. Elution

30. Chromatogram

31. Fraction

32. Chromatogram patterns

33. Elution profiles

34. FPLC

35. Elutionvolume

36. Diffusion of molecules with matrix

37. Resolution

38. FPLCsystem

39. FPLCcolumn

40. Column types

41. FPLC system

42. Column packing

43. Sample preparation

44. Buffer

45. Washing

46. Gradient

47. Chromatogram in FPLC page

48. Chromatogram profiles troubleshooting

49. Referencespage

1. High Performance Liquid Chromatography (Figure 1) and Fast Protein Liquid Chromatography

- This is a tool to separate compounds according to its hydrophobicity or hydrophilicity.
- A gradient is given from hydrophilic to hydrophobic range of solvent moving through a solid matrix.
- Here are some valuable tips collected through experience and handed over so that the reader can find it useful when the chromatograms and the instruments prove so cross with you.
- The instrumentation and working for both the systems HPLC and FPLC are almost same.
- HPLC is High Performance/Pressure Liquid Chromatography(Figure 2) and FPLC is Fast Protein Liquid Chromtography. (Figure 3)

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Figure 1 HPLC (Source:www.Wikipedia.com)

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Figure 2 HPLC flowpath

Concepts

Aquaintance with a few concepts are essential to know the functioning of HPLC

2. Beer lambert law

- Beer lamberts law explains the calculation of concentration in the spectrophotometer's flow cell (Figure 11)
- Beers law is abbreviated as A = Ecl
- The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as:

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where A is the measured absorbance, a(lambda) is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as:

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where epsilon is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1.

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Figure 11 Flow cell , a is absorption, b is pathlength, c is concentration of solute

3. Theoretical Plates

- Separation of the solute happens based on the number of theoretical plates of matrix.
- The number of theoretical plates can be calculated from the bead size of the column and from the length of the column.
- Alternatively it can also be calculated from the peak width and the height.
- Theoretical plates are the imaginary plates of size the thickness of one bead size.
- So one theroretical plate is a plate consisting of a single layer of beads of silica particles. From this, the number of theoretical plates running throughout the column can be calculated from the length ofthe column.
- The use of calculating this is to directly determine the efficiency of the column.
- The more the number of theoretical plates, the more the efficiency.
- This is because the sample gets more distance to get separated out by flowing for a longer distance and it diffuses more laterally depending on the flow rate.
- The lower the flow rate, the higher the efficiency of diffusion and more the binding of the sample to the matrix.
- The higher the flowrate, the higher is the resolution or the sharpness of the peak, but with lesser the substance eluted.
- Theoretical plate calculation needs length ofthe column in nm and size ofthe silica beads in nm.
- Beads of silica are arranged as imaginary stacked plates, each plate consisting of one single layer of beads.
- The number of theoretical plates will be the number of stacked imaginary plates, each plate having single layer of silica beads.
- The more the number of theoretical plates the more will be separation.
- The nature of silica beads causes separation. So, each plate of silica bead has a imaginaray constant ofseparation capacity. (Figure 20)

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Figure 20 A single layer of imaginary silica bead with a defined separation capacity is one theoretical plate

4. Calculation of Theoretical Plates

- Half Peak Height Method (www.shimadzu.com)
- Width is calculated from the width at half the peak height (W0.5). Since width can be calculated easily by hand, it is the most widely used method.
- N, the number of theoretical plates, is one index used to determine the performance and effectiveness of columns, and is calculated using equation, where tr: retention time, and W: peak width

- N = 5.54(tr/W0.5)²

5. Resolution

- Resolution is the ability of the column to separate between two closely lying objects in terms of their elution percentage.
- If two compounds are separated by a marginal differnces in their elution time or the percentage of elution, the ability to better distinguish between them, by achieving two separate peaks, that are separated by a larger baseline determines how good the samples are separated.
- To better resolve clustered peaks is to use a resolved gradient or a step gradient.
- Resolved gradient uses the concept of first having run a trace and then running a gradient that is focussed only to that particular cluster of peaks to be resolved next.
- Alternatively, step gradient can be used by change in flow rate at the areas where there are clusters.
- The gradient goes in steps when seen in the direction offlow.
- The flow rate decreases with a prolonged time at points of peaks. When it comes to a flat baseline, the flow rate increases with decreased time.
- Elution time is the time at which the sustance elutes out.
- It is the same as retention time, the time after which the sample is no longer retained in the column.
- The general term used is the retention time, abbreviated RT.
- Retention factor. Retention time calculation. (Figure 10)
- Concentration of the eluted compound can be calculated by noting the AU, absorption unit ofthe solute.
- There are different limits set for AU of detection.
- Most commonly used are 1.5 to 2 AU for the lab usage and research purposes.
- Its simply that we dont inject concentrations beyond a limit into a column which may affect the stability of the column.
- Accordingly to suit a wide range of columns, analytical, semi preparative and preparative we set the elution high maxima ofthe peak as 2.56 AU.
- Something more than this is not very precise and at this range disturbance is less.
- Necessary dilution of the sample, if concentrated, is a must to allow for the optimal falling ofthe range ofthe peaks well below 2 AU.
- So by noting the AU of one peak of the chromatogram and taking the respective collected peak's UV-VIS absorption profile or concentration in a spectrophotometer, the concentration ofthe remaining peaks in the chromatogram can be labelled too.

6. Phases

- HPLC is a gradient system that makes use of the concept of adhering the solute in a preequilibriated column and then passing a gradient to elute each compound at its specific solubility in the compound which depends on its polarity.
- The extraction ofthe compound happens between different phases.
- There are basically two phases namely Mobile phase and Stationary phase.
- The solvent from the equilibriation is adhered to the column as stationary phase. It exchanges for solutes from the mobile phase.
- There is a stationary phase of solvent adhered to the matrix of the column.
- This stationary phase can be made to be either of the solvents used in the gradient having two opposite polarities.
- Partitioning separation is based on the analyte's relative solubility between two liquid phases( Figure 25 )
- Bonded Phases in the column vary as follows:- Silanol -Si-OH

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Figure 25 Partitioning separation is based on the analyte's relative solubility between two liquid phases

7. Calculation of Retention Time and Absorbance

- One observational value from the chromatogram is needed to proceed with calulation of retention time and absorbance of peaks.
- The start point of a chromatogram is 0 time, and the end point is, say 60 minutes
- The X axis of the chromatogram is the Retention time value.
- Divide the X axis into half from between the start and stop time points. This will be halfthe value, 30 minutes
- Similarly divide and mark the X axis and read the corresponding value of RT of the Peaks
- From the tip of the peak, a vertical line perpendicular to the X axis is drawn, the point this meets the X axis is the RT..
- Y axis is absorbance value in AU (just termed as Absorbance Unit).
- Note the peaks elution absorbanc unit value from the detector screen.
- Use this AU value of the peak to graduate the remaining points on the Y axis.
- The concentration of this peak is found by reading the value in a UV-VIS spectrophotometry

8. Column capacity

- Overloading a column can cause poor peak shapes and other problems. Column capacity depends on many factors, but typical values are:(Supelco bulletin 826)
- Semi-preparative column (25cm x 10mm) <100mg
- Preparative column (25cm x 21.2mm) < 500mg
- Analytical column (25cm x 4.6mm) <500pg

9. Components

- The components of a HPLC are pumps, matrix in a column, detector, recorder, controller, gradient mixer, gaurd column, injector and a fraction collector.
- There are two solvent tanks having hydrophilic solvent in one and a hydrophobic solvent in another.
- These two are taken into the pumps by means oftubings fitted with a filtering frits.
- Materials used in downstream
- VarianCary 100 Bio UV-Visible Spectrophotometer, CONDA laboratories, Madrid, Spain.),UV/Vis detector SPD-20A (Shimadzu Corporation, Kyoto, Japan)
- HPLC Controller 2152 LKB Bromma, HPLC Pump 2150 LKB Bromma, Analytical Agilent Zorbax 300SB-C18 (5micron RP Column 14*25mm)
- Solvents (Analytical Rasayan, India), HPLC 100% pure methanol , Milli Q water, all chemicals used were of analytical grade purity.
- Merck Analytical Grade TLC plates

10. Pumps

- The pumps are capable of delivering very high pressure.
- It is comparable to a lorry weight.
- The pumps are two stroke.
- They have sapphire rods that pump the solvents and is turned by a shaft that is connected to a electric motor.
- The flow of solvents through the two stroke is controlled by a flow compensating mechanism that interchanges the inlets of solvents between the two stroke pumps.
- Pump A is for MilliQ water. Its enough to program pump В from 5% to 95%. Pump В is for Acetonitrile. Add 0.1 % TFA to Acetonitrile.
- To pass solvents through HPLC column, we needs high pressure.
- The pressure is generated by electric motors driving sapphire rods that pump the incoming solvent with high pressure equalling a ton.
- A two stroke pumps are there for each solvent Pulse compensation is necessary to keep a constant flow from the two stroke pumps.
- There is no gap in the solvents passed through this way compared to a normal two stroke.
- The idea is to pass a part of the the outlet of the first stroke pump into the inlet of the second stroke pump and the vice versa.
- This way, a gapless non turbulent flow is obtained in the combined outlet of the two stroke pump.
- The solvents from the tanks travel through frits and then into the respective pumps, from where a gradient is created by the controller and gets mixed in a gradient mixer, from where only one outlet arises and enters the injector.
- Once the solute is injected the injector, it travels through the loop along with the gradient set by the controller. It enters the gaurd column, then the column, then it enters the flow cell of the UV VIS detector and then flows into the fraction collector and discard.

11. Flowpath

- The solvent inlet of the pump A is connected to the hydrophilic solvent and the hydrophobic solvent is connected as per convention.
- So the percentage of the gradient set is in terms of percentage B.
- The inlets are passed through titanium frits to avoid the entry of the dust present in the solvent.
- The outlet of each the pump is connected with a junction to a mixer, hence called as the gradient mixer. (Figure 22)
- The outlet of the gradient mixer is connected to the injector of the sample.
- The injector is also connected to the inlet of the column.
- The column is protected by a gaurd column just to avoid the entry of larger particles than the poresize.
- The gaurd column has a cartridge that can be replaced every once in a while. The outlet of the column is connected to the UV-VIS detector.
- The UV-VIS detector has a flow cell, halogen and deuterium lamps for detection in full range ofthe spectrum.
- The oultet of the UV- VIS detector is connected to the fraction collector.
- The detector is also connected to the recorder of the chromatogram.
- The chromatogram is a graph that shows the elution of compounds as peaks with the Y axis showing the intensity or absorbance unit and the X axis showing the retention or elution time.

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Figure 21 A ferrule connects tubings airtightly to a column

12. Tubings

- Its suggested to keep all the tubings either metal or solvent resistant PVCs.
- Tubings are to be kept minimal distance in joining the systems so that the values of elu- tion and the purity of the collected fractions good.
- A ferrule connects tubings airtightly to a column (Figure 21)

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Figure 22 Pump A with two stokes connected to solvent A and gradient mixer.

13. Leak

- Fixing a leak near the ferrules is done by unscrewing the nut of the ferrule with a spanner a little bit and then tightening it back to fix the leak.
- It does not work to just tighten the screw and stop the leak.
- In higher leaks the nut is removed and changed for a newer ferrule, in slow flowing conditions of the solvent, to prevent any air from entering the pump.
- Pump leaks are common.
- The flow rate of both the pumps have to be checked for the same constant flow.
- Incase one pump has a higher flow rate than the other, the gradient may not be proper.
- It will also show a fluctuating wavy pattern in the chromatogram.
- Most outside leaks happen due to displa]cement of ferrules, a conical metal attached to the end of solvent tubes in the hplc.
- The ferrules prevent the leakage of solvent through the gap at the junctions of the machine parts and the tube ends.
- So, the ferrules are made to stick into cones of the machinery into which the tubes end. So, such leaks can be cured by just loosening the concerned knob, and retightening to allow the screwing thread to sit proper.
- Negative peaks result in the initial phase ofthe chromatogram due to the dilution of the sensitive gradient flowing into the column by the extra addition of solvent from the sample that is dissolved in it.
- If joints are giving leaks, then unscrew by spanner and rotate the pipe and then rescrew this in order to seal the gap.
- A set of spanners with all sizes used for the nuts and bolts of HPLC is usually a handy thing that is supplied with the instrument and proves indispensable at times of repair.
- Spare replacements and tubings come along with the instrument and knowledge on replacing these in repair is essential to be very good in hplc.
- During all these replacements or repair to clean, it is advisable to keep the run of the solvent always on at a low flow rate so that, air does not enter.
- Changing ferrule can be done in case there is excessive leak even after tightening the joints. The ferrule of the right size is fixed onto the steel or pressure plastic pipe.
- A 2 or 3mm of free wire is left in front of the ferrule tip to fix into the socket properly.
- If the ferrule is stuck into the socket then it is pullet out with the help of a syringe needle.

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