1. HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY(HPTLC)
(In the name of GOD)
HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY(HPTLC)
Dr. A.R.Bekhradnia

2. HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY(HPTLC)
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3. THIN LAYER CHROMATOGRAPHY (TLC)
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4. CHROMATOGRAPHY
Chromatography is a physical process of separation in which the components to be separated are distributed between 2 immiscible phases ­ a stationary phase which has a large surface area and mobile phase which is in constant motion through the stationary phase.
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5. THIN LAYER CHROMATOGRAPHY (TLC)
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6. Mikhail Tsvet Born 14 May 1872 Asti, Italy Died 26 June 1919 (age 47)
Nationality Russia
Fields botany
Mikhail Semyonovich Tsvet (Михаи́л Семёнович Цвет, also spelled Tsvett, Tswett, Tswet, Zwet, and Cvet) (1872–1919) was a Russian-Italian botanist who invented adsorption chromatography.
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7. Invention of Chromatography by M. Tswett
Ether
Chromatography
Colors
Chlorophyll
CaCO3
The Russian-Polish botanist M. Tswett is generally recognized as the first person to establish the principles of chromatography.
In a paper he presented in 1906, Tswett described how he filled a glass tube with chalk powder (CaCO3) and, by allowing an ether solution of chlorophyll to flow through the chalk, separated the chlorophyll into layers of different colors. He called this technique “chromatography”.
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8. Comparing Chromatography to the Flow of a River...
Light leaf
Heavy stone
Water flow
Chromatography can be often compared to the flow of a river.
A river consists of a stationary riverbed and water that continuously moves in one direction. What happens if a leaf and a stone are thrown into the river? The relatively light leaf does not sink to the bottom, and is carried downstream by the current. On the other hand, the relatively heavy stone sinks to the bottom, and although it is gradually pulled downstream by the current, it moves much more slowly than the leaf.
If you stand watch at the mouth of the river, you will eventually be able to observe the arrival of the leaf and the stone. However, although the leaf will arrive in an extremely short time, the stone will take much longer to arrive.
This analogy represents the components of chromatography in the following way:
River: Separation field
Leaf and stone: Target components of sample
Standing watch at the river mouth: Detector
Base
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9. Chromato-graphy / -graph / -gram / -grapher
Chromatography: Analytical technique
Chromatograph: Instrument
Chromatogram: Obtained “picture”
Chromatographer: Person
There are many similar terms in this field and so let us clarify some of them.
“Chromatography” is the name of the analytical technique itself.
A “chromatograph” is an analytical instrument that is used to perform chromatography. The product names of the chromatographs given in the catalogs of analytical instrument manufacturers should all include this word.
A “chromatogram” is produced by recording the results obtained with chromatography on recording paper (or some other medium).
A “chromatographer” is a person who carries out a chromatography experiment.
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10. Three States of Matter and Chromatography Types
Mobile phase
Gas
Liquid
Solid
Stationary phase
Gas chromatography
Liquid chromatography
There are various ways of categorizing chromatography. Here, let us categorize it in terms of the three states of matter.
There are generally three states of matter: gas, liquid, and solid. If we could use stationary phases and mobile phases of any state, this would give a total of nine different types of chromatography. Using a gas as the stationary phase or a solid as the mobile phase, however, is not practical (even if it is possible) and this restricts the combinations that can be used.
Chromatography performed using a gas as the mobile phase and a liquid or a solid as the stationary phase is called “gas chromatography” (GC). Chromatography performed using a liquid as the mobile phase and a liquid or a solid as the stationary phase is called “liquid chromatography” (LC). Both of these techniques are indispensable, particularly in the field of organic chemistry.
In addition to these, there is a technique called “supercritical fluid chromatography” (SFC), in which a supercritical fluid kept at a high temperature and high pressure is used as the mobile phase.
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11. 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.
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12. Interaction Between Solutes, Stationary Phase, and Mobile Phase
Differences in the interactions between the solutes and stationary and mobile phases enable separation.
Solute
Degree of adsorption, solubility, ionicity, etc.
The solutes interact with the stationary and mobile phases. These interactions are the most important contributing factor behind separation.
Representative examples of the types of interactions that take place in liquid chromatography are given below. (They are not based on strict classifications.)
Adsorption
Distribution
Hydrophobic interaction
Ion exchange
Ion pair formation
Osmosis and exclusion
Affinity
Stationary phase
Mobile phase
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13. Classification According to the force of separation:
Adsorption chromatography
Partition chromatography
Ion exchange chromatography
Gel filtration chromatography
Affinity chromatography
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14. Column Chromatography and Planar Chromatography
Separation column
Paper or a substrate coated with particles
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
Column Chromatography
Paper Chromatography
Thin Layer Chromatography (TLC)
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15. Separation Process and Chromatogram for Column Chromatography
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”.
Output concentration
Chromatogram
Time
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16. Chromatogram tR tR : Retention time t0 t0 : Non-retention time h
Peak
Intensity of detector signal t0
t0 : Non-retention time h
A : Peak area A
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.
h : Peak height
Time
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17. Separation Process and Chromatogram for Column Chromatography
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”.
Output concentration
Chromatogram
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Time

18. THIN LAYER CHROMATOGRAPHY
Once the solvent is within ~1-2 cm of the top of the TLC sheet, the TLC is removed from the developing chamber and the farthest extent of the solvent (the solvent front) is marked with a pencil.
The solvent is allowed to evaporate from the TLC sheet in the hood.
The spots are visualized using a UV lamp.
A fluorescent compound, usually Manganese-activated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will fluoresce light green by itself, but spots of analyte quench this fluorescence and appear as a dark spot.
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19. THIN LAYER CHROMATOGRAPHY - Visualization  
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
Visualization of spots under a UV254 lamp. The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.
Iodine vapors are a general unspecific color.
Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate.
Once visible, the Rf value of each spot can be determined
Chromatogram of 10 essential oils,
Stained with vanillin reagent.
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20. THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
The Rf is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin)
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21. THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
The Rf is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin)
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22. THIN LAYER CHROMATOGRAPHY – Rf’s
Rf values can be used to aid in the identification of a substance by comparison to standards.
The Rf value is not a physical constant, and comparison should be made only between spots on the same sheet, run at the same time.
Two substances that have the same Rf value may be identical; those with different Rf values are not identical.
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23. THIN LAYER CHROMATOGRAPHY – Rf’s
Absorption of Solutes
The adsorption strength of compounds increases with increasing polarity of functional groups, as shown below:
-CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR2, -CO2H.
(weakly adsorbed) (strongly adsorbed)
(nonpolar) (more polar)
Elution Strength of Mobile Phase (e)
Elution strength is generally considered to be equivalent to polarity. A solvents elution strength depends on Intermolecular Forces between the solvent and the analytes and between the solvent and the stationary phase.
A more polar (or more strongly eluting solvent) will move all of the analytes to a greater extent, than a less polar, weakly elution solvent.
For example, the elution strength of hexane is very low; e = 0.01.
the elution strength of ethyl acetate is higher; e = 0.45
the elution strength of ethanol is even higher; e = 0.68
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24. Solvent Properties and Elution Strengths
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25. Elution Strength of Mixed Solvents
The elution strength of the mixture is assumed to be the weighted average of the elution strengths of the components:
eonet = eoA (mole % A) + eoB (mole % B)
where: mole % A = (moles A) / (moles A + moles B)
Thus, to determine the eonet of a solvent mixture, the molar ratio of the solvents must first be calculated. For example, the eonet of a solvent mixture prepared from 1.0 mL of ethyl acetate plus 9.0 mL of hexanes is calculated as shown below:
eonet = eoEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] +
eohexane [(moles hexane)/(moles EtOAc+moles hexane)]
where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc]
thus: eonet = {0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]}
{(1.0 mLEtOAc)(0.902g/mL)/88.11g/mole) + (9.0 mLhexane)(0.659g/mL)/86.18g/mole)}
and eonet =
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26. Resolution
The separation between two analytes on a chromatogram can be expressed as the resolution, Rs and can be determined using the following equation:
Rs = (distance between center of spots) (average diameter of spots)
In TLC, if the Rs value is greater than 1.0, the analytes are considered to be resolved. x x
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27. Improving Resolution:
For two closely migrating components, optimum resolutions are usually obtained when the Rf’s of both compounds are between 0.2 and 0.5
* To Improve Rs, change the elution strength of the solvent to optimize Rf’s
change eonet, all compounds will be effected similarly.
Alter the composition of the solvent system so that the components affinity for the mobile phase vs. the solid phase are differentially changed (= change in selectivity).
Changing the chemical nature of the solvent system, such as changing a hydrogen bonding solvent to a solvent which cannot hydrogen bond to the analyte, is often the most effective.
** Improve Rs by decreasing the diameter of the analyte spots. This can be achieved by applying smaller and less concentrated spots.
TLC/TLCprocedure.html
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28. HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY(HPTLC)
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29. Introduction of HPTLC
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way.
HPTLC takes place in high­speed capillary flow range of the mobile phase.
There are three main steps HPTLC procedure, they are
1] Sample preparation, volume precision and exact position are achieved by use of suitable instrument.
2] Solvent (mobile phase) migrates the planned distance in layer (stationary phase) by capillary action. In this process sample separated into it’s components.
3] Separation tracks are scanned in densitometer with light
beams in visible or uv region
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30. Steps Involving in HPTLC
Selection of chromatography layer
Sample Preparation
Pre-washing
Pre-conditioning
Application of sample
Chromatography development
Detection of spots
Scanning & documentation
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31. Selection of chromatography layer
Sample preparation
Normal phase chromatography: non polar solvent
Reversed phase chromatography: polar solvent
Selection of chromatography layer
Depends on nature of material to be separated
Commonly used(silica gel, alumina)
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32. It is purification step Mainly methanol is used
Pre-washing
It is purification step
Mainly methanol is used
Essential for quantitative evaluation
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33. Linomat lV applicator
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34. Dr. A.R.Bekhradnia

35. Selection of HPTLC plates
Previously hand made plates is used in TLC for both qualitative and quantitative work. Certain drawbacks with that is non­uniform layer, formation of thick layer, paved for advent of precoated plates.
Nowadays precoated plates are available in different format and thickness by various manufactures. Precaoted plates can be used for both qualitative and quantitative work in HPTLC, they are
GLASS PLATES
POLY ESTER/POLYETHYLYNE
ALUMINIUM PLATES
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36. Glass Plates: - fragile - high weight - higher production cost
Offers superior flat and smooth surface.
- fragile
- high weight
- higher production cost
Polyester/polyethylene plates:
Thickness of plate is 0.2mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packing material is required.
Development of plate cann’t be above temperature
1200 c loses its shape.
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37. Aluminium plates: - Thickness of plate is 0.1mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packaging material is required.
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38. SORBENTS USED IN HPTLC PLATES:
sorbents which are used in convential TLC are also used in HPTLC with or without modification.
- silica gel 65F
- highly purified silicagel 60
- aluminium oxide
- cellulose microcrystalline
- silica gel
- reversed stationary phase
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39. Layer thickness
The layer thickness in HPTLC is around cm,in conventional it is 250mm.
Layer prewashing:
- Ascending method
- Dipping method
- Continuous method
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40. ACTIVATION OF PRECOATED PLATES
The plates are activated by placing in an oven at 110­1200 C for 30 min, this step will removes water that has been physically absorbed on surface at solvent layer.
Freshly opened box of HPTLC plates usually does not require activation.
Activation at higher temp and for longer time is avoided which leads to very active layer and there is risk of sample being decomposed.
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41. Solvents used in HPTLC - Methanol (commonly used)
- Chloroform:methanol:ammonia(90:10:1)
- Chloroform:methanol(1:1)
- Methylene chloride:methanol(1:1)
- Ammonia(1%)solution
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42. Application of sample and standard
Usual concentration range is 0.1-1µg / µl,above this causes poor separation.
Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe on TLC plates as bands.
Band wise application - better separation - high response to densitometer.
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43. Processes in the Developing Chamber
The «classical» way of developing a chromatogram is to place the plate in a chamber, which contains a sufficient amount of developing solvent. The lower end of the plate should be immersed several millimeters. Driven by capillary action the developing solvent moves up the layer until the desired running distance is reached and chromatography is stopped. The following considerations primarily concern silica gel as stationary phase and developments, which can be described as adsorption chromatography.
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44. Provided the chamber is closed, four partially competing processes occur:
Between the components of the developing solvent and their vapor, an equilibrium will be established eventually (1). This equilibrium is called chamber saturation. Depending on the vapor pressure of the individual components the composition of the gas phase can differ significantly from that of the developing solvent.
While still dry, the stationary phase adsorbs molecules from the gas phase. This process, adsorptive saturation, is also approaching an equilibrium in which the polar components will be withdrawn from the gas phase and loaded onto the surface of the stationary phase (2).
Simultaneously the part of the layer which is already wetted with mobile phase interacts with the gas phase. Thereby especially the less polar components of the liquid are released into in the gas phase (3). Unlike (1) this process is not as much governed by vapor pressure as by adsorption forces.
During migration, the components of the mobile phase can be separated by the stationary phase under certain conditions, causing the formation of secondary fronts.
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45. NANOMAT
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46. AUTOMATIC TLC SAMPLER
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47. CHROMATOGRAM DEVELOPMENT
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48. Ancillaries for Chromatogram Development in a tank
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49. Flat Bottom Chamber
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50. Pre-equilibration Also called Chamber Saturation
Low polarity mob. Phase:- no need
High polar mob. Phase:- desirable
For reverse phase saturate chamber with polar solvent
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51. LIGHT WEIGHT TWIN TROUGH CHAMBER
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52. Twin Trough Chamber pre-equilibration with solvent vapor
               
pre-equilibration with solvent vapor
               
LOW SOLVENT CONSUMPTION
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53. CAMAG Twin Trough Chambers offer several ways to improve the results of TLC/HPTLC developing techniques. It allows low solvent consumption, reproducible pre-equilibration with solvent vapor, equilibration performed with any liquid and for any period of time, and development is started only when developing solvent is introduced into the trough with the plate. 
Twin Trough Chambers are available with stainless steel lid or as a Light-Weight Twin Trough Chamber made from highly transparent sheet glass with a glass lid.
               
Start of development
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54. Chromatographic development and drying
After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere.
Dry in vacuum desiccator - avoid hair drier because essential oil components may evaporate.
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55. Automatic Developing Chamber ADC
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56. Detection and visualization
Detection under UV light is first choice - non destructive.
Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length).
Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF.
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57. UV –INSPECTION -UV Lamps.
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58. DENSITOMETRIC CHROMATOGRAM EVALUATION
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59. TLC Scanner 3 with “CATS” Soft Ware.
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60. CATS STANDARD PROGRAM.
CATS PROGRAM OPTIONS
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61. Video Scan.
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62. Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution.
When individual component does not respond to UV - derivatisation required for detection .
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63. HPTLC TLC 100µm 250µm High due to smaller particle size generated Less
Shorter migration distance and the analysis time is greatly reduced
Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes
New type that require less amount of mobile phase
Auto sampler
Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer
TLC
250µm
Less cm
Slower
Silica gel , Alumina
More amount
Manual spotting
Not possible
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64. APPLICATIONS Pharmaceutical Researches Bio­medical Analysis
Clinical Analysis
Environmental Analysis
Food Industry
Therapeutic drug monitoring to determine concentration of drug
and it’s metabolite in blood, urine etc
Analysis of environmental pollutions levels
Quantitative determination of prostaglandin’s and thromboxanes
in plasma
Analysis of nitrosoamines in food and body fluids
Determination of sorbic acid in wine
Characterization of hazards in industrial waste
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