1. LIQUID CHROMATOGRAPHY
CHAPTER 21 pg. 604
Adsorption Chromatography

2. Introduction
The principle of liquid chromatography is a separation process based on distribution between two phases, where the sample components is propelled by liquid which percolates a solid stationary phase.

3. Liquid Chromatography
Chromatography in which the mobile phase is a liquid.
The liquid used as the mobile phase is called the “eluent”.
The stationary phase is usually a solid or a liquid.
In general, it is possible to analyze any substance that can be stably dissolved in the mobile phase.
“Liquid chromatography” (LC) is chromatography in which the mobile phase is a liquid.
Stationary Phase
Usually a solid or a liquid is used as the mobile phase. (This includes the case where a substance regarded as a liquid is chemically bonded, or applied, to the surface of a solid.)
The most common form of stationary phase consists of fine particles of, for example, silica gel or resin packed into a cylindrical tube. These packed particles are called “packing material” or “packing” and the separation tube into which they are packed is called the “separation column” or simply the “column”. In day-to-day analysis work, “column” is sometimes used to refer to the stationary phase and “stationary phase” is sometimes used to refer to the column.
Mobile Phase
Various solvents are used as mobile phases. The mobile phase conveys the components of the dissolved sample through the separation field, and facilitates the repeated three-way interactions that take place between the phases and the sample, thereby leading to separation.
The solvent used for the mobile phase is called the “eluent” or “eluant”. (In LC, the term “mobile phase” is also used to refer to this solvent. In this text, however, we shall use the term “eluent”.)
Sample
In general, it is possible to analyze any substance that can be stably dissolved in the eluent. This is one advantage that LC has over GC, which cannot be used to analyze substances that do not vaporize or that are thermally decomposed easily.
The sample is generally converted to liquid form before being introduced to the system. It contains various solutes. The target substances (the analytes) are separated and detected.

4. Column Chromatography and Planar Chromatography
Separation column
Column Chromatography
Paper or a substrate coated with particles
Paper Chromatography
Thin Layer Chromatography (TLC)
Liquid chromatography can be categorized by shape of separation field into column-shaped and planar types.
A representative type of chromatography that uses a column-shaped field is “column chromatography”, which is performed using a separation column consisting of a cylindrical tube filled with packing material. Another type is “capillary chromatography”, which is performed using a narrow hollow tube. Unlike column chromatography, however, capillary chromatography has yet to attain general acceptance. (In the field of GC, however, capillary chromatography is a commonly used technique.)
Types of chromatography that use a planar (or plate layer) field include “thin layer chromatography”, in which the stationary phase consists of a substrate of glass or some other material to which minute particles are applied, and “paper chromatography”, in which the stationary phase consists of cellulose filter paper.
Packing material

5. Separation Process and Chromatogram for Column Chromatography
Output concentration
Time
Chromatogram
The separation process for column chromatography is shown in the above diagram.
After the eluent is allowed to flow into the top of the column, it flows down through the spaces in the packing material due to gravity and capillary action. In this state, a sample mixture is placed at the top of the column. The solutes in the sample undergo various interactions with the solid and mobile phases, splitting up into solutes that descend quickly together with the mobile phase and solutes that adsorb to the stationary phase and descend slowly, so differences in the speed of motion emerge. At the outlet, the elution of the various solutes at different times is observed.
A detector that can measure the concentrations of the solutes in the eluate is set up at the column outlet, and variations in the concentration are monitored. The graph representing the results using the horizontal axis for times and the vertical axis for solute concentrations (or more accurately, output values of detector signals proportional to solute concentrations) is called a “chromatogram”.

6. Chromatogram tR : Retention time t0 : Non-retention time A : Peak area
Intensity of detector signal
Peak
tR : Retention time h A
t0 : Non-retention time
A : Peak area
h : Peak height
Usually, during the time period in which the sample components are not eluted, a straight line running parallel to the time axis is drawn. This is called the “baseline”.
When a component is eluted, a response is obtained from the detector, and a raised section appears on the baseline. This is called a “peak”. The components in the sample are dispersed by the repeated interactions with the stationary and mobile phases, so the peaks generally take the bell-shape form of a Gaussian distribution.
The time that elapses between sample injection and the appearance of the top of the peak is called the “retention time”. If the analytical conditions are the same, the same substance always gives the same retention time. Therefore, the retention time provides a means to perform the qualitative analysis of substances.
The time taken for solutes in the sample to go straight through the column together with the mobile phase, without interacting with the stationary phase, and to be eluted is denoted as “t0”. There is no specific name for this parameter, but terms such as “non-retention time” and “hold-up time” seem to be commonly used.
Because the eluent usually passes through the column at a constant flow rate, tR and t0 are sometimes multiplied by the eluent flow rate and handled as volumes. The volume corresponding to the retention time is called the “retention volume” and is notated as VR.
The length of a straight line drawn from the top of a peak down to the baseline is called the “peak height”, and the area of the raised section above the baseline is called the “peak area”.
If the intensities of the detector signals are proportional to the concentrations or absolute quantities of the peak components, then the peak areas and heights are proportional to the concentrations of the peak components. Therefore, the peak areas and heights provide a means to perform the quantitative analysis of sample components. It is generally said that using the peak areas gives greater accuracy.
Time

7. From Liquid Chromatography to High Performance Liquid Chromatography
Higher degree of separation!  Refinement of packing material (3 to 10 µm)
Reduction of analysis time!  Delivery of eluent by pump  Demand for special equipment that can withstand high pressures
The arrival of high performance liquid chromatography!
In order to increase the separation capability of column chromatography, in addition to increasing the surface area of the stationary phase so that the interaction efficiency is increased, it is also necessary to homogenize the separation field as much as possible so that dispersion in the mobile and stationary phases is minimized. The most effective way of achieving this is to refine the packing material.
Refining the packing material, however, causes resistance to the delivery of the eluent to increase. This is similar to the way that water drains easily through sand, which has relatively large particles, whereas it does not drain easily through clay-rich soil, which has relatively fine particles.
Depending on gravity and capillary action would cause analysis to take a very long time to be completed, and the idea of delivering the eluent forcibly using a high-pressure pump was proposed. This was the start of high performance liquid chromatography.

8. Flow Channel Diagram for High Performance Liquid Chromatograph
Pump
Sample injection unit
(injector)
Column
Column oven
(thermostatic column chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
A high performance liquid chromatograph differs from a column chromatograph in that it is subject to the following performance requirements.
Solvent Delivery Pump
A solvent delivery pump that can maintain a constant, non-pulsating flow of solvent at a high pressure against the resistance of the column is required.
Sample Injection Unit
There is a high level of pressure between the pump and the column; a device that can inject specific amounts of sample under such conditions is required.
Column
The technology for filling the column evenly with refined packing material is required. Also, a material that can withstand high pressures, such as stainless steel, is required for the housing.
Detector
Higher degrees of separation have increased the need for high-sensitivity detection, and levels of sensitivity and stability that can respond to this need are required in the detector.
Degasser

9. Detection in HPLC *There are six major HPLC detectors:
Refractive Index (RI) Detector
Evaporative Light Scattering Detector (ELSD)
UV/VIS Absorption Detectors
The Fluorescence Detector
Electrochemical Detectors (ECDs)
Conductivity Detector
* The type of detector utilized depends on the characteristics of the analyte of interest.

10. Refractive Index Detector
Based on the principle that every transparent substance will slow the speed of light passing through it.
Results in the bending of light as it passes to another material of different density.
Refractive index = how much the light is bent
The presence of analyte molecules in the mobile phase will generally change its RI by an amount almost
linearly proportional to its
concentrations.

11. Refractive Index Detector
Affected by slight changes in mobile phase composition and temperature.
Universal-based on a property of the mobile phase
It is used for analytes which give no response with other more sensitive and selective detectors.
RI = general
responds to the presence of all solutes in the mobile phase.
Reference= mobile phase
Sample= column effluent
Detector measures the differences between the RI of the reference and the sample.

12. Evaporative Light Scattering Detector (ELSD)
Analyte particles don’t scatter light when dissolved in a liquid mobile phase.
Three steps:
1) Nebulize the mobile phase effluent into droplets.
Passes through a needle and mixes with hydrogen gas.
2) Evaporate each of these droplets.
Leaves behind a small particle of nonvolatile analyte
3) Light scattering
Sample particles pass through a cell and scatter light from a laser beam which is detected and generates a signal.

13. UV/VIS Absorption Detectors
Different compounds will absorb different amounts of light in the UV and visible regions.
A beam of UV light is shined through the analyte after it is eluted from the column.
A detector is positioned on the opposite side which can measure how much light is absorbed and transmitted.
The amount of light absorbed will depend on the amount of the compound that is passing through the beam.

14. UV/VIS Absorption Detectors
Beer-Lambert law: A=εbc
absorbance is proportional to the compound concentration.
Fixed Wavelength: measures at one wavelength, usually 254 nm
Variable Wavelength: measures at one wavelength at a time, but can detect over a wide range of wavelengths
Diode Array Detector (DAD): measures a spectrum of wavelengths simultaneously

15. The Fluorescence Detector
Measure the ability of a compound to absorb then re-emit light at given wavelengths
Some compounds will absorb specific wavelengths of light which, raising it to a higher energy state.
When the compound returns to its ground state, it will release a specific wavelength of light which can be detected.
Not all compounds can fluoresce / more selective than UV/VIS detection.
fluorescence.jpg

16. Electrochemical Detectors (ECDs)
Used for compounds that undergo oxidation/reduction reactions.
Detector measures the current resulting from an oxidation/reduction reaction of the analyte at a suitable electrode.
Current level is directly proportional to the concentration of analyte present.
Conductivity Detector:
Records how the mobile phase conductivity changes as different sample components are eluted from the column.

17. Types
Exclusion
Ion Exchange
Affinity
Hydrophobic interaction

18. 1. Size Exclusion Chromatography
Sample separated based on size.
Stationary phase has specific pore sizes.
Larger molecules elute quickly.
Smaller molecules penetrate inside the pores of the stationary phase and elute later.

19. 2. Ion Exchange Chromatography
Used with ionic or ionizable samples.
Stationary phase has a charged surface.
opposite charge to the sample ions
Cation exchange resin: sulfonic acid or –COOH
Anion exchange resin: tertiary or primary amine
The mobile phase = aqueous buffer
The stronger the charge on the analyte, the more it will be attracted to the stationary phase, the slower it will elute.

20. 3. Affinity Chromatography
Stationary phase: covalently bonding affinity ligands to a solid support (agarose, porous glass bead)
Affinity ligands: antibodies, enzyme inhibitors, molecules that reversibly and selectively bind to analytes
Analyte molecules are retained by the stationary phase change the mobile phase to release the analyte
Mobile phase: 1. support strong bonding between analyte and the ligands. 2. eliminate the analyte ligand interaction. Change of pH or μ
A: Extraordinary specificity
Application: rapid isolation of biomolecules

21. 4. Hydrophobic Interaction Chromatography

22. Instruments
HPLC
FPLC

23. HPLC

24. HPLC Chromatogram

25. FPLC

26. FPLC Chromatogram

27. Stationary phase
The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is silica gel, followed by alumina. Cellulose powder has often been used in the past. Also possible are ion exchange chromatography, reversed-phase chromatography(RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, though in EBA a fluidized bed is used. There is an important ratio between the stationary phase weight and the dry weight of the analyte mixture that can be applied onto the column. For silica column chromatography, this ratio lies within 20:1 to 100:1, depending on how close to each other the analyte components are being eluted

28. Mobile phase (eluent)
The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the retention factor value of the compound of interest is roughly around in order to minimize the time and the amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer chromatography (TLC) with the same stationary phase.
There is an optimum flow rate for each particular separation. A faster flow rate of the eluent minimizes the time required to run a column and thereby minimizes diffusion, resulting in a better separation. However, the maximum flow rate is limited because a finite time is required for analyte to equilibrate between stationary phase and mobile phase, see Van Deemter's equation. A simple laboratory column runs by gravity flow. The flow rate of such a column can be increased by extending the fresh eluent filled column above the top of the stationary phase or decreased by the tap controls. Faster flow rates can be achieved by using a pump or by using compressed gas (e.g. air, nitrogen, or argon) to push the solvent through the column (flash column chromatography).
The particle size of the stationary phase is generally finer in flash column chromatography than in gravity column chromatography. For example, one of the most widely used silica gel grades in the former technique is mesh 230 – 400 (40 – 63 µm), while the latter technique typically requires mesh 70 – 230 (63 – 200 µm) silica gel.
A spreadsheet that assists in the successful development of flash columns has been developed. The spreadsheet estimates the retention volume and band volume of analytes, the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted

29. Column Chromatogram Resolution Calculation
Typically, column chromatography is set up with peristaltic pumps, flowing buffers and the solution sample through the top of the column. The solutions and buffers pass through the column where a fraction collector at the end of the column setup collects the eluted samples. Prior to the fraction collection, the samples that are eluted from the column pass through a detector such as a spectrophotometer or mass spectrometer so that the concentration of the separated samples in the sample solution mixture can be determined.
For example, if you were to separate two different proteins with different binding capacities to the column from a solution sample, a good type of detector would be a spectrophotometer using a wavelength of 280 nm. The higher the concentration of protein that passes through the eluted solution through the column, the higher the absorbance of that wavelength.

30. Because the column chromatography has a constant flow of eluted solution passing through the detector at varying concentrations, the detector must plot the concentration of the eluted sample over a course of time. This plot of sample concentration versus time is called a chromatogram.
The ultimate goal of chromatography is to separate different components from a solution mixture. The resolution expresses the extent of separation between the components from the mixture. The higher the resolution of the chromatogram, the better the extent of separation of the samples the column gives. This data is a good way of determining the column’s separation properties of that particular sample. The resolution can be calculated from the chromatogram.
The separate curves in the diagram represent different sample elution concentration profiles over time based on their affinity to the column resin. To calculate resolution, the retention time and curve width are required.

31. Retention Time: The time from the start of signal detection by the detector to the peak height of the elution concentration profile of each different sample.
Curve Width: The width of the concentration profile curve of the different samples in the chromatogram in units of time.
A simplified method of calculating chromatogram resolution is to use the plate model. The plate model assumes that the column can be divided into a certain number of sections, or plates and the mass balance can be calculated for each individual plate. This approach approximates a typical chromatogram curve as a Gaussian distribution curve. By doing this, the curve width is estimated as 4 times the standard deviation of the curve, 4σ. The retention time is the time from the start of signal detection to the time of the peak height of the Gaussian curve.

32. Rs = 2(tRB – tRA)/(wB + wA)
From the variables in the figure above, the resolution, plate number, and plate height of the column plate model can be calculated using the equations:
Resolution (Rs)
Rs = 2(tRB – tRA)/(wB + wA)
Where:
tRB = retention time of solute B tRA = retention time of solute A wB = Gaussian curve width of solute B wA = Gaussian curve width of solute A Plate Number (N): N = (tR)2/(w/4)2 Plate Height (H): H = L/N Where L is the length of the column.