Faculty of Pharmacy, Comenius University in Bratislava

Laboratory Practices from Pharmaceutical Chemistry

JIŘÍ KOS VLADIMÍR GARAJ 2020 Comenius University in Bratislava

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NH

NH

OO

O

CH3

CH3

N

NH

O

NH2

NH

NCH2

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ONH

OH

Cl

NO2

Cl

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© Authors:

Jiří Kos [3,49 AH], Vladimír Garaj [3,49 AH]

Faculty of Pharmacy at Comenius University in Bratislava

reviewer: prof. PharmDr. Josef Jampílek, Ph.D.

The authors are responsible for the professional and linguistic side.

First edition, Bratislava, 2020

© Comenius University in Bratislava

ISBN 978-80-223-4947-5

This work is licensed under a Creative Commons Attribution 4.0 International License.

https://creativecommons.org/licenses/by/3.0/

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Table of Contents

Introduction ............................................................................................................................................ 8

1. Laboratory safety rules ................................................................................................................... 9

2. Basic Laboratory Equipment: ........................................................................................................ 13

3. Basic approaches to pharmaceutical analysis ............................................................................... 19

3.1. Determination of melting point ............................................................................................ 19

3.1.2. Principle ........................................................................................................................ 19

3.1.3. Instrumentation ............................................................................................................ 20

3.2. Surface activity ...................................................................................................................... 21

3.2.1. Adsorption at the mobile phase interface .................................................................... 21

3.3. Ultraviolet-visible spectroscopy ............................................................................................ 22

3.3.1. Principle ........................................................................................................................ 22

3.3.2. Instrumentation ............................................................................................................ 22

3.4. Infrared spectroscopy ........................................................................................................... 23

3.4.1. Principle ........................................................................................................................ 23

3.4.2. Instrumentation ............................................................................................................ 24

3.5. Thin layer chromatography ................................................................................................... 25

3.5.1. Principle ........................................................................................................................ 25

3.5.2. Instrumentation ............................................................................................................ 26

3.6. High performance liquid chromatography ........................................................................... 26

3.6.1. Principle ........................................................................................................................ 26

3.6.2. Instrumentation ............................................................................................................ 27

3.7. NMR spectroscopy ................................................................................................................ 30

3.7.1 General information ...................................................................................................... 30

3.7.2 1H NMR spectroscopy ................................................................................................... 33

3.7.3 13C NMR spectroscopy .................................................................................................. 39

4. Procedures for drug synthesis and evaluation of their structure, purity, stability and

physicochemical properties .......................................................................................................... 43

4.1. ACETYLSALICYLIC ACID .......................................................................................................... 43

4.1.1. Synthesis ....................................................................................................................... 43

4.1.2. Mechanism of reaction ................................................................................................. 44

4.1.3. Solubility ........................................................................................................................ 45

4

4.1.4. Melting point ................................................................................................................. 45

4.1.5. Chromatography ........................................................................................................... 45

4.1.6. Surface tension ............................................................................................................. 46

4.1.7. Spectroscopy ................................................................................................................. 46

4.1.8. pKa Evaluation ............................................................................................................... 50

4.1.9. Acetylsalicylic acid stability evaluation ......................................................................... 51

4.2. PARACETAMOL ...................................................................................................................... 51

4.2.1. Synthesis ....................................................................................................................... 51

4.2.2. Synthesis of phenacetin ................................................................................................ 52

4.2.3. Solubility ........................................................................................................................ 53

4.2.4. Melting point ................................................................................................................. 53

4.2.5. Chromatography ........................................................................................................... 54

4.2.6. Surface tension ............................................................................................................. 54

4.2.7. Spectroscopy ................................................................................................................. 55

4.2.8. Partition coefficient ...................................................................................................... 58

4.2.9. Accelerated paracetamol stability evaluation .............................................................. 59

4.3. BENZOCAINE ......................................................................................................................... 61

4.3.1. Synthesis ....................................................................................................................... 61

4.3.2. Solubility ........................................................................................................................ 62

4.3.3. Melting point ................................................................................................................. 62

4.3.4. Chromatography ........................................................................................................... 63

4.3.5. Surface tension ............................................................................................................. 63

4.3.6. Spectroscopy ................................................................................................................. 63

4.3.7. Partition coefficient ...................................................................................................... 68

4.4. ESTERS OF 4-HYDROXYBENZOIC ACID (PARABENS) .............................................................. 69

4.4.1. Synthesis ....................................................................................................................... 69

4.4.2. Solubility ........................................................................................................................ 70

4.4.3. Melting point ................................................................................................................. 70

4.4.4. Chromatography ........................................................................................................... 71

4.4.5. Surface tension ............................................................................................................. 71

4.4.6. Spectroscopy ................................................................................................................. 72

4.4.7. Partition coefficient ...................................................................................................... 86

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4.5. DISULFIRAM .......................................................................................................................... 87

4.5.1. Synthesis ....................................................................................................................... 87

4.5.2. Solubility ........................................................................................................................ 88

4.5.3. Melting point ................................................................................................................. 88

4.5.4. Chromatography ........................................................................................................... 89

4.5.5. Surface tension ............................................................................................................. 89

4.5.6. Spectroscopy ................................................................................................................. 90

4.5.7. Partion coefficient ......................................................................................................... 94

4.6. CAFFEINE ............................................................................................................................... 96

4.6.1. Synthesis ....................................................................................................................... 96

4.6.2. Solubility ........................................................................................................................ 98

4.6.3. Melting point ................................................................................................................. 98

4.6.4. Chromatography ........................................................................................................... 99

4.6.5. Spectroscopy ................................................................................................................. 99

4.7. TRIMECAINE ........................................................................................................................ 103

4.7.1. Synthesis ..................................................................................................................... 103

4.7.2. Solubility ...................................................................................................................... 105

4.7.3. Melting point ............................................................................................................... 105

4.7.4. pKa Evaluation ............................................................................................................. 106

4.7.5. Chromatography ......................................................................................................... 106

4.7.6. Spectroscopy ............................................................................................................... 106

4.7.7. Partition coefficient .................................................................................................... 111

4.7.8. Accelerated trimecaine stability evaluation ............................................................... 112

4.8. LIDOCAINE ........................................................................................................................... 114

4.8.1. Synthesis ..................................................................................................................... 114

4.8.2. Solubility ...................................................................................................................... 116

4.8.3. Melting point ............................................................................................................... 116

4.8.4. pKa Evaluation ............................................................................................................. 117

4.8.5. Chromatography ......................................................................................................... 117

4.8.6. Spectroscopy ............................................................................................................... 117

4.8.7. Partition coefficient .................................................................................................... 122

4.9. PHENYTOIN ......................................................................................................................... 123

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4.9.1. Synthesis ..................................................................................................................... 123

4.9.2. Solubility ...................................................................................................................... 125

4.9.3. Melting point ............................................................................................................... 125

4.9.4. Surface tension ........................................................................................................... 126

4.9.5. Chromatography ......................................................................................................... 126

4.9.6. Spectroscopy ............................................................................................................... 126

4.9.7. Partition coefficient .................................................................................................... 130

4.10. BARBITAL ......................................................................................................................... 131

4.10.1. Synthesis ..................................................................................................................... 131

4.10.2. Solubility ...................................................................................................................... 133

4.10.3. Melting point ............................................................................................................... 133

4.10.4. Chromatography ......................................................................................................... 133

4.10.5. Spectroscopy ............................................................................................................... 133

4.11. SULFANILAMIDE .............................................................................................................. 137

4.11.1. Synthesis ..................................................................................................................... 137

4.11.2. Solubility ...................................................................................................................... 140

4.11.3. Melting point ............................................................................................................... 140

4.11.4. Surface tension ........................................................................................................... 140

4.11.5. Chromatography ......................................................................................................... 141

4.11.6. Spectroscopy ............................................................................................................... 141

4.11.7. Partition coefficient .................................................................................................... 145

4.12. DIPROPHYLLINE ............................................................................................................... 146

4.12.1. Synthesis ..................................................................................................................... 146

4.12.2. Solubility ...................................................................................................................... 147

4.12.3. Melting point ............................................................................................................... 147

4.12.4. Surface tension ........................................................................................................... 147

4.12.5. Chromatography ......................................................................................................... 148

4.12.6. Spectroscopy ............................................................................................................... 148

4.12.7. Partition coefficient .................................................................................................... 152

4.12.8. Accelerated diprophylline stability evaluation ........................................................... 153

4.13. ISONIAZID ........................................................................................................................ 156

4.13.1. Synthesis ..................................................................................................................... 156

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4.13.2. Solubility ...................................................................................................................... 157

4.13.3. Melting point ............................................................................................................... 157

4.13.4. Chromatography ......................................................................................................... 158

4.14.5. Spectroscopy ............................................................................................................... 158

5. Microwave synthesis ................................................................................................................... 161

5.1. Principle of heating by microwave radiation ...................................................................... 162

5.1.1. Loss angle .................................................................................................................... 163

5.1.2. Relaxation time ........................................................................................................... 165

5.2. Techniques in microwave synthesis .................................................................................... 165

5.3. Microwave synthesis of niclosamide .................................................................................. 166

5.3.1. Synthesis ..................................................................................................................... 166

5.3.2. Solubility ...................................................................................................................... 166

5.3.3. Melting point ............................................................................................................... 167

5.3.4. Chromatography ......................................................................................................... 167

6. IR Spectrum Table & Chart .......................................................................................................... 168

7. NMR Chemical Shifts of Impurities Charts .................................................................................. 178

8. Common Solvents Used in Organic Chemistry - Table of properties .......................................... 182

9. References ................................................................................................................................... 183

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Introduction The synthesis of a new molecule plays a key role in the drug discovery process. A drug

candidate results from a lengthy and intense research activity, where many molecules are synthesized and extensively tested. The intense research requires scientists with creativity, a vast range of scientific knowledge, and great persistence. The presented study text is a basic material intended for practical teaching of the master's program of the subject Pharmaceutical Chemistry at the Faculty of Pharmacy at Comenius University, where students can have light experience with this inevitable step of a drug discovery process. The first part of the text is focused on basic approaches to pharmaceutical analysis (melting point, ultraviolet-visible spectroscopy, Infrared spectroscopy, thin layer chromatography, high-performance liquid chromatography, and NMR spectroscopy). The process of the individual syntheses is clearly described and explained by the reaction schemes in the second part of the text. The final synthesis products are characterized by available spectral methods (IR, 1H and 13C NMR). The text includes procedures for determining the basic physico-chemical parameters (melting point, solubility, surface activity, acid-base properties, partition coefficient, thin layer chromatography - TLC, stability tests and reaction kinetics parameters) of the resulting products.

Our thanks go to doc. Mgr. Fils Andriamainty, PhD.; Ing. Tomáš Pekárek, Ph.D. and Mgr. Branislav Horváth, PhD.

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1. Laboratory safety rules While working in the laboratory for pharmaceutical chemistry, you will be exposed

to numerous chemical and physical hazards. Tasks in chemistry require a long time and tiredness after 8 hours of focusing on the work in the laboratory increases the risk of unfortunate events even more. Such events may lead sometime to harmful repercussions or even dead. To minimize the risk, it is necessary to acquire the following rules:

1. Follow the instructions! Whether it's listening to your teacher or lab technician or following a procedure in a book, it's critical to listen, pay attention, and be familiar with all the steps, from start to finish, before you begin. If you are unclear about any point or have questions, get them answered before starting, even if it's a question about a step later on in the protocol. Know how to use all of the lab equipment before you begin. This might be the most important rule. By not following it you can ruin your experiment, but more importantly, endanger yourself, your colleagues, and damage the equipment.

2. Each accident, however minor, is reported to your supervisor. By notifying your supervisor, even if no action is taken, the incident must be recorded.

Personal protection safety rules and dress code

1. You should always wear a lab coat in the laboratory. 2. When working with equipment, hazardous materials, glassware, heat, and/or

chemicals, always wear face shields or safety glasses. Splash hazards are perhaps the most significant danger present in the lab, and eyes are extremely sensitive.

3. Wear goggles instead of contact lenses. Contact lenses can absorb chemicals from the air, concentrate and hold them against the eye, and prevent proper flushing of the eye should a chemical be splashed into the eyes.

4. Never wear sandals or other open-toed shoes in the lab. Footwear should always cover the foot completely. High heels are not permitted in the laboratory because of your balance.

5. Never wear shorts or skirts in the lab. The reason is again to protect your skin from direct exposure to the chemical if a spill occurs.

6. Do not wear jewelry. Remove rings, dangling earrings, and necklaces. 7. Do not wear clothes that hang, such as loose sleeves, neckties, or scarfs. These

pose fire hazards as well as chemical hazards. 8. Long hair is to be constrained for the same reason as aforementioned. 9. When handling any toxic or hazardous agent, always wear the latex gloves. 10. When using lab equipment and chemicals, be sure to keep your hands away from

your face (mouth, eyes). 11. Never eat, drink, or chew gum in the laboratory. Neither keep the food or

beverages in the working area. There is too much risk of contaminating your food. 12. Do not apply makeup (including lip balm and other lip balms) in the lab. 13. Before leaving the laboratory or eating, always wash your hands with soap. 14. Do not use a phone nor listen to radios, MP3 player or any other devices of this

type in the laboratory.

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Safety Equipment

1. Know the Location of Safety Equipment. Make sure you know where your lab's safety equipment (including fire extinguishers, first aid kit, safety showers and emergency power shut down switch) is located and how to properly use it.

2. Make sure you are aware of where your lab's exits and fire alarms are located.

Housekeeping safety rules

1. Always keep your work area tidy, uncluttered, and clean. Only the materials you require for your work should be kept in your work area. Everything else should be stored safely out of the way. Remove from the bench glassware, reagents, and equipment that you no longer need to avoid cramming workspaces with unnecessary items. This not only helps you work more efficiently but also reduces the chances of knocking over items causing breakages or spillages.

2. Ensure that items like stools and bags do not block paths and aisles to reduce tripping hazards. This is especially crucial in case of an emergency and people need to evacuate fast.

3. Close your lab drawer. Once you have retrieved the equipment you need from your equipment drawer, be sure to close it again. Open drawers can pose tripping hazards (especially bottom drawers) and obstruction of walkways. Also, don’t let the key in the lock of the drawer to prevent it from accidental bending and breaking. It is better to put the key to the drawer.

Chemical safety rules

1. Use chemicals only as directed and for their intended purpose. Unauthorized experiments or procedures must not be attempted.

2. Never smell or taste chemicals. 3. Never use mouth suction to fill a pipette. Use a pipette bulb or other suitable

device. 4. Before removing any of the contents from a chemical bottle, read the label

twice. 5. Never put a dropper into a reagent bottle. Instead, put the reagent in a beaker

so you can bring it back to your desk and use a dropper there. 6. Do not take more chemicals from a bottle than you need for your work. 7. Do not put unused chemicals back into the stock container. 8. If a chemical spill occurs, clean it up right away. Be especially careful of spills

around the balances. These electronic devices are sensitive to corrosion. If needed, ask the laboratory technician for a brush to clean the balance.

9. Should a chemical spill on your person, immediately remove all affected clothing (tops from the back forward to avoid dragging the chemical across your face) and wash the affected body area with copious amounts of water. If a large portion of your clothing is affected with strong acid or alkali, immediately get to the safety shower and remove the contaminated clothing while the water is running.

10. Do not dispose of chemicals down the drain. Most chemicals must be disposed of as hazardous waste.

11. Water should not be poured into concentrated acid. Instead, pour acid slowly into the water while stirring constantly. In many cases, mixing an acid with water is exothermic.

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Laboratory Equipment

1. Each time you use glassware, be sure to check it for chips and cracks. Heating defective glassware can cause that glassware to break (or explode!), resulting in a spill. Notify your lab technician of any damaged glassware.

2. Never pick up broken glassware with your bare hands, regardless of the size of the pieces. A brush and dustpan are provided for broken glassware. Never put broken glass in a regular garbage can. A container is provided that is specially designed for broken glassware.

3. If an instrument or piece of equipment fails during use or isn't operating properly, report the issue to a technician right away.

General Guidelines

1. In the case of pregnancy, the supervisor must be informed. Exposure to certain chemicals may adversely affect the developing fetus during pregnancy. If you have any medical condition which you think may adversely affect your ability to safely perform in the lab, or that makes you particularly at risk to be in the lab, please inform your supervisor.

2. Many items (glass, metal, etc.) look exactly the same hot they do cool. Be very careful whenever heating something that all of your equipment (beakers, flasks, ring stands, etc.) are cool before handling them.

3. In the event of a chemical splashing into your eyes or on your skin, immediately flush the affected area with running water for at least 10 minutes. In the case of acids on the skin, wash also with bicarbonate solution and finally with water. In the case of alkalis on the skin, wash also with a solution of citric or acetic acid and finally with water.

4. Never leave an ongoing experiment unattended.

5. If you are not feeling well, report it to the laboratory supervisor immediately.

Fire

Using organic solvents combined with heating creates potential for flash fire, explosion, rapid spread of fire, and high toxicity of products of combustion. Small bench-top fires in lab spaces are typical and not uncommon.

1. In the case of fire, push the button of emergency power shut down switch.

2. If a fire should occur in a beaker or some other container, cover it with a glass dish or other nonflammable item. Never move any object that is burning and never use water to extinguish a chemical fire.

3. If a small portion of your clothes catches fire, the fire may be extinguished by patting it out. If a larger portion of your clothes should catch fire, either drop to the ground and roll or use the safety shower. Never use a fire extinguisher on a person.

4. If a fire is large enough to warrant the use of a fire extinguisher, the proper use of the extinguisher is as follows:

1. Pull the Pin.

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2. Aim extinguisher nozzle at the base of the flames.

3. Squeeze trigger while holding the extinguisher upright.

4. Sweep the extinguisher from side to side, covering the area of the fire with the extinguishing agent.

Extinguishers are only for use on relatively small fires. Duration of the blast is, depending on the size, 10 seconds to 30 seconds of spray. Keep yourself between the fire and your exit. If the fire is too big don't try to fight it.

Laboratory accidents and first aid Thermal burns:

Run the burned area under cool tap water with low pressure for 10-15 minutes. Then let the laboratory technician do the first aid.

Chemical burns:

Remove any clothing contaminated by the chemical, if it is not stuck to the skin. Rinse the skin with plenty of gently running water for 10-15 minutes. Wrap the burned area with a gauze dressing or a clean piece of cloth. In the case of acids on the skin, wash also with saturated bicarbonate solution and finally with water. In the case of alkalis on the skin, wash also with 1% citric acid and finally with water.

Electrical burns:

Stop the source of electricity or remove the injured person from the source of electricity using a plastic or wooden object. Evaluate breathing and pulse. If there is no pulse, cardiopulmonary resuscitation should be started.

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2. Basic Laboratory Equipment: Basic laboratory equipment can be classified as follows:

Glassware

o Volumetric and graduated

“To contain” - Flask will have precisely the volume listed inside it.

[measuring cylinders and volumetric flasks]

“To deliver” - Precisely the volume listed will leave it when the contents

drain out of the vessel. The drainage holdback error has already been

considered during calibration [calibrated pipettes and burets]

o Boiling and reaction

o Other (technical)

Porcelain and plastic ware

Metal tools

When a measured liquid is placed in a volumetric glass the surface takes on a curved shape. This curve is known as a meniscus. Volumetric glassware is calibrated such that reading the bottom of the meniscus, when it is viewed at eye level, will give accurate results. Viewing the meniscus at any other angle will give inaccurate results.

The accuracy of the markings on volumetric glassware varies greatly. The markings on beakers and flasks are usually about plus or minus 5% of the volume of the container. As such, they should be used only when a rough estimate of the volume is required. The tolerance for graduated cylinders is about 1%. Volumetric flasks, burets and pipets are the most accurate with tolerances of less than 0.2%. To achieve these accuracies the person using the device needs to use the proper technique and the measurements need to be made at the temperature for which the glassware was calibrated (usually 20 °C).

Pipette:

A pipette (Figure 1) is used for accurate measurement of small volumes. A graduate pipette can be used to measure any volume up to the maximum of the respective pipette, the scale is usually numbered from top to bottom so that the value 0 is at the top. Volumetric pipettes can only be used for one volume, but they are more accurate, the measure is indicated by a thin line. When using a pipette, start by holding the pipette vertically near the top so it is easy to get your index finger over the top. Squeeze the air out of the rubber bulb and place the bulb on the burette top. Place the burette into the liquid to be drawn up and slowly release the pressure on the bulb, allowing the vacuum created to suck up the liquid. Once the liquid is above the calibration mark, take the pipette bulb off and cap the pipette with your index finger. Slowly allow the liquid to flow out of the pipette until the bottom of the meniscus is right at the calibration mark. Once the fluid is at the correct level, lift the pipette out of the fluid, and touch the tip to the side of the container to get any excess drops off. Put the pipette over the container you want the liquid in and take your finger off of the top

of the pipette. Holding the pipette vertically, allow the fluid to flow out on its own without

Figure 1. Pipette

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trying to force the liquid out. Once the flow stops, touch the tip of the pipette to the side of the container to get any last drops off and remove the pipette.

Burette:

Burrete (Figure 2a) is used during titration, its tap discharges the reagent solution from it into the titration flask, according to the scale on the burette we can determine the exact volume of reagent used.

When filling the burette with reagent, check the valve to be sure it is closed. Pour the reagent from a beaker into the top of the burette. A funnel may be used if necessary. As you are filling the burette, at some point, pause and look at the tip to be sure the liquid is not pouring out of the bottom. If it is, take necessary action immediately to contain the reagent and clean up the spill. Fill the burette slightly above the “zero” mark. Remove the funnel from the top of the burette, put a waste beaker under the burette tip and open the burette tip to set the level of the reagent to the “zero” mark and by this, you also expel the air and fill

the tip with the reagent. Some burrets (Figure 2b) are set directly on a stock bottle, when the titration solution

is too dangerous (e.g. perchloric acid), and these are filed using a rubber bulb while sealing a small opening in the glass, where the bulb is connected, by an index finger. Do not make higher pressure inside the stock bottle than is necessary for pushing the titration solvent up to the burette! Very high pressure will cause shooting the burette from the stock bottle instead of speeding up the filling of burette.

Volumetric flask:

the most accurate measurement of a larger volume (calibrated

to four significant figures),

with a characteristic shape with a long neck, on which there is a

line indicating the liquid level of a given volume,

are calibrated to refill, they can only measure the volume

marked on them,

are very accurate and are intended for volume measurement

only.

If you are dissolving materials in the flask, once you have placed in your reagent, do not fill the flask to the graduation mark initially. Instead, fill the bulb about half full; this will allow you to swirl more vigorously to get the solid to dissolve. If you want to shake the flask, put the stopper on it first. If the solid does not dissolve

immediately, add a little more water and continue. Once the solid has dissolved completely,

Figure 2. Burette (a) and automatic burrete (b) (Source: https://www.duran-

group.com/en/products-solutions/laboratory-

glassware/products/volumetric-glassware/burette.html

Figure 3. Volumetric flask

a b

15

fill the flask (Figure 3) to the graduation mark. There should not be any liquid over the line indicating volume for accurate volume measurement. Graduated cylinders:

The graduated cylinders (Figure 4) are wider glass cylindrical containers allowing measurement of liquid volume. The accuracy of volume measurement depends on the size of the measuring cylinders, they are of different sizes (10, 25, 50, 100, 250, 500, 1000, 2000 ml). Most graduated cylinders are accurate to three significant figures (as opposed to flasks and beakers that are accurate to only two significant figures, and burettes and pipettes that are accurate to four significant figures). Always look past the wall of the glass and read the volume at the center of the liquid. The graduated

cylinders cannot be heated or allowed to react.

Test tubes:

The simplest and most common devices of various types have a

cylindrical shape

Used to hold and mix liquids for chemical analysis or

microreactions

Beaker: Beaker (Figure 6) is the most used chemical glass. It is used only for approximate volume measurement but has a more universal use (we can pour solutions in them, mix liquids, heat them, ...).

Figure 4. Graduated cylindres

Figure 5. Test tubes

Figure 6. Beakers

16

Reaction flask:

Used for boiling or reactions

Single- / double- / triple-neck

Flat-bottom / round-bottom

Only a round-bottomed flask with one neck can be used for vacuum distillation, eg when removing the solvent on a vacuum evaporator.

Erlenmayer flask:

Erlenmayer flask (Figure 8) is used for:

mixing substances

heating liquids

storing substances

sometimes is calibrated and can serve to

measure the indicative volume similarly as a beaker

Condensers:

Reflux condenser – intended to return the liquid

toward the source of the vapor to prevent the

evaporation of solvent during heating.

Condenser for distillation - receive the vapor

through one opening and deliver the liquid through

opposite opening

Liebig Allihn Grahams

Figure 7. Reaction flasks

Figure 8. Erlenmayer flasks

Figure 9. Condensers

17

Funnel:

A funnel (Figure 10a) can be

used for pouring liquids into

containers with narrow necks,

or for normal filtration.

Büchner funnel (Figure 10b) is a

fritted/perforated funnel used

for the vacuum-assisted

filtration together with side-

arm flask.

Separatory funnel (Figure 10c) is used to separate layers of immiscible liquids. They are

commonly for liquid-liquid extractions, separating mixture′s components into two

solvent phases of different densities. They have a stopcock at the bottom, which can be

opened or closed to drain liquid, as well as a tap at the top that can be opened to release

excess vapor pressure.

Dropping funnels (Figure 10d) are used for adding liquids into reaction mixture by

deploying on double- or triple-neck reaction flask. A stopcock allows the flow to be

controlled. They are often graduated.

Desiccator: Desiccator (Figure 11) is used for drying substances or to avert get wet already dried hygroscopic material. It contains a desiccant (lumps of silica gel, freshly calcined quicklime, anhydrous calcium sulfate, or anhydrous calcium chloride) at the bottom. A stopcock on a vacuum desiccator allows the desiccator to be evacuated.

Figure 10. Funnels

Figure 11. Desiccator

a b c d

18

Watch glass / petri dish:

Watch glass (Figure 12) is usually used for weighing of, as a cover for

a beaker or evaporating a small amount of liquid.

Petri dish (Figure 13) is used in a chemical lab

in similar way.

Evaporating dish:

An evaporating dish (Figure 14) is

used to evaporate excess solvents

to produce a concentrated solution

or a solid precipitate of the

dissolved substance.

Mortar and pestle:

Mortar and pestle (Figure 15) are used for crushing and grinding

substances into a fine powder or paste.

Wash bottle:

A wash bottle (Figure 16) is a squeeze bottle with a nozzle, that is used

to add solvent or rinse various pieces of laboratory glassware.

Figure 12. Watch glass

Figure 13. Petri dish

Figure 14. Evaporating dish

Figure 15. Mortar and pestle

Figure 16. Wash bottle

19

3. Basic approaches to pharmaceutical analysis

3.1. Determination of melting point

3.1.2. Principle

The state of matter of a substance is, among other things, conditioned by temperature and pressure. Normal melting point is referred to as the melting point which was measured at normal pressure, i.e. 101.325 kPa [in older literature, older units such as bar and torr were used (1 bar = 105 Pa, 1 torr = 133.322 Pa)].

The heated solid substance changes to liquid state when the melting point is reached. For each pure crystalline chemical substance, the melting point is characteristic (melts at a certain temperature). Crystalline substances can occur in different crystal modifications/systems (atoms can be arranged in different crystal lattices). We can talk about so-called polymorphism of substances. Each polymorph of the same substance has a different melting point depending on the type of crystal modification. In addition to other instrumental methods (e.g. X-ray analysis), the melting point or various methods of thermal analysis can also be used to determine the type of crystal modification. It should be mentioned that, that the melting point is also affected by the type of solvent used in the crystallization.

Amorphous substances (without crystal lattice, e.g. glass or paraffin) do not have a characteristic melting point. When heated, they gradually soften until they turn into a liquid state.

By increasing the internal energy of substances, for example by heating, their temperature increases, which can be reflected in a change of state. As the temperature increases, particles in crystal lattice oscillate faster, increasing the amplitude of their deflection. When the melting point is reached, the crystal lattice begins to disintegrate, and the particles become freely moving. Substances whose particles are more tightly bound in the crystal lattice have a higher melting point than substances whose particles are less tightly bound. The heat (energy) supplied to the crystalline substance heated to the melting point is fully utilized to overcome the bonding forces, until the crystal lattice completely disintegrates. This does not increase the kinetic energy, but the potential energy of particles. Chemical pure crystalline substance heated to the melting point does not increase its temperature during further heating until all of it melts (it is necessary to ensure uniform heating of the entire volume tested substance).

During melting, most substances increase in volume because the particles arranged in the crystal lattice are generally closer to each other than in liquid state. An increase in external pressure contributes to an increase in the forces that hold these particles together (therefore, for most substances, the melting temperature increases with increasing pressure). Several substances that reduce their volume during melting are an exception, such as water. In this case, increased pressure helps to maintain the liquid state. At higher pressure, ice melts bellow 0 °C.

The melting point for a given substance is the criterion of its purity respectively demonstrating the correct/desired polymorph. Most pure substances melt in the range of 0.5 °C. The presence of impurities and moisture usually results in a decrease of the melting point.

20

3.1.3. Instrumentation

Determination of melting point in a capillary

To determinate the melting point of a sample of a substance placed in capillary, either glass thermoblock (e.g. Thiele, Roth thermoblock) or a metal block in the shape of a prism or a cylinder with a cavity for thermometer and a sight glass for observing the substance in the capillary is used (Figure 17). The block is made of metal that conducts heat well (e.g. aluminium, brass). The required capillaries should have a diameter of a 1 mm, a wall thickness of a 0,15-0,20 mm, and a length of about 50 mm. The capillary is sealed at one end. With the non-sealed end of the capillary, we gradually collect enough of the scattered sample of the substance to form a continuous column 2-3 mm high after shaking off. When determining the temperature in the glass thermoblock, we attach the capillary to the thermometer so that the sample is placed at the level of the mercury tank. When determining the temperature in a metal block, we choose the length of the capillary so that we can remove it from the block without any problems.

Figure 17. Thermoblock (Source: https://www.thermofisher.cz/produkty/bodotavek-smp-11)

After reaching a temperature about 20 °C lower than the expected melting point, the

heating rate should not exceed 3 °C per minute. The melting point is the temperature range in which the column of material in the capillary collapses at the beginning and the last remnants of the crystal in the melt disappear at the end. The narrower is this range, the cleaner is the measured substance and the higher is the proportion of one polymorph.

If it is a substance whose melting point is unknown, we first perform an orientation measurement. We increase temperature faster and as soon as the crystalline structure of the substance begins to collapse, we stop the heating and the obtained value is considered as an approximate melting point. After the cooldown of a measurement device, we will continue the determination with the correct procedure.

Determination of melting point under a microscope

The determination of the melting point under a microscope is performed on a so-called microheating bench (e.g. Kofler’s bench) (Figure 18). The basic part of this device is an electrically heated block on/in which, depending on the type, we place the sample as a microscopic specimen in a glass. The melting process of the crystals is monitored by the ocular and the melting temperature is recorded. The device also includes equipment for temperature regulation and melting. The heating speed is automatically pre-set.

21

Figure 18. Kofler’s bench

3.2. Surface activity

One of the physicochemical properties that affects the effect of the drug is the ability to adsorb at the phase interface. This adsorption is due to the effect of unbalanced forces at the phase interface. Depending on the nature of the phases, we distinguish between adsorption at the mobile phase interface (gas-liquid phase, or two immiscible liquid phases) and adsorption at the solid phase interface. Due to the focus of this textbook, we will focus only on adsorption at the mobile phase interface.

3.2.1. Adsorption at the mobile phase interface

If we examine the phase interface formed by the liquid and gas phases, we find that the liquid tends to obtain a shape with the smallest possible surface (spherical shape) at a given volume. This shape is given by the action of forces, when greater attractive intermolecular forces act from the liquid environment than from the side of the gas phase (air). The result is the drawing of surface molecules into the liquid phase, the molecules in the surface layer being attracted by molecules below the surface. This surface tension is a

characteristic feature of all liquids. The surface tension of liquids () is the force that acts on the surface of a liquid perpendicular to the unit of length of the surface. The unit is the newton per meter (N.m-1). Its size is inversely proportional to temperature.

If we dissolve the substance in a liquid, two situations can occur in terms of adsorption: Negative adsorption - The adhesive forces between the solvent and solute molecules

are greater than the forces between the solute molecules, then the solute molecules will be drawn from the surface into the volume.

Positive adsorption - The adhesive forces between the solvent molecules and the solute are less than the forces between the solute molecules, the solute molecules will be expelled from the solution and will collect on the surface.

Surfactants have the ability to reduce surface tension. They have a higher concentration in the surface layer than inside the liquid. At the mobile phase interface, substances which are surfactants in a given solvent are adsorbed. Surface activity is related to the polarity of molecules. At the water-oil phase interface, amphiphilic molecules that have a polar hydrophilic group (-OH, -NH2, -COOH, -NO2, -COH- etc.) and at the same time a non-polar hydrophobic group (hydrocarbon chain) show surface activity. Examples are fatty acids, alcohols or soaps. The surface activity increases in a given homologous series with the extension of the hydrocarbon chain until it reaches a certain maximum. Subsequently, it decreases again as the solubility in the aqueous medium also decreases significantly. It is logical that water-insoluble substances cannot accumulate at the phase interface. Surface activity can be quantified by measuring surface tension. We can measure it directly (tear-off

22

method using torsional weights) or indirectly (measurement of capillary elevation or depression or drop method using a stalagmometer).

3.3. Ultraviolet-visible spectroscopy

3.3.1. Principle

Ultraviolet (UV) and visible (VIS) spectrophotometry is an optical method based on the absorption of radiation by a substance at wavelengths of 200-400 nm and 400-800 nm respectively. The absorption of UV-VIS radiation by the molecule causes a transition of valence electrons from the ground to excited states.

The grouping of atoms in a molecule where radiation is absorbed is called the chromophore. For organic compounds, the absorption of radiation is conditioned by the transition of electrons between binding (π) or non-binding (n) molecular orbitals to anti-binding (π* or σ*). In aromatic or heterocyclic compounds, extensive conjugate bonds systems (ππ* transitions) or free electron pair of heteroatoms – N, O, S (nπ* or nσ* transitions) serves as chromophores. Increasing the conjugation shifts the absorption to higher wavelengths up to the visible light region. As chromophores in non-aromatic compounds contain multiple bonds, groups such as C=C, C=O, C=N, N=N, N=O absorb radiation. Out of the inorganic substances, they can absorb complex compounds with transition metal ions in the UV-VIS region

The value of the wavelength in the maximum absorption of the absorption band (λmax) determines the difference in the energy of the ground and the excited state.

The absorption intensity, i.e. the value of the molar absorption coefficient (εmax) of the absorption band, is related to the probability with which transition will take place. UV-VIS spectrophotometry is used mainly for quantitative analysis, unlike infrared spectroscopy, and also for qualitative analysis – identification (based on the shape of the spectrum position of absorbtion maxima and the position of the analyte peak in the chromatogram, see below in HPLC section. The most used method of substance detection is based on the principle of spectrophotometry because it excels especially in simplicity and sensitivity.

Qualitative analysis, i.e. identification of the substance, is most often performed by comparing the absorption spectra (number and position of absorption maxima) of the standard and the test sample. It should be noted that UV spectrometry, unlike infrared spectroscopy, is used primarily for quantitative analysis.

Quantitative analysis, i.e. the determination of the substance content, is based on the validity of the Lambert-Beer law. The determination is performed at the maximum wavelength of the absorption band of the analyte. The determination of the sample concentration is usually performed by the calibration method of the standard solution. If the calibration is linear and reproducible, linear regression is performed and the actual calculation of the sample content is performed on the basis of the determined parameters of the straight-line equation.

3.3.2. Instrumentation

Photometers or spectrophotometers which allow working with monochromatic light are used to measure UV-VIS absorption. Each spectrophotometer consists of:

Source of radiation – tungsten lamp and hydrogen or deuterium lamp

23

Monochromator – spectral filters, prism, grating,

Cuvettes – quartz

Detector – photocells, photomultipliers

Measuring device - galvanometer

Nowadays, measurements are most often performed on two-beam spectrophotometers. Two-beam spectrophotometers are automatic devices in which the monochromatic beam is optically divided into two identical beams. One passes through the sample cuvette, the other through the blank solution cuvette. The resulting intensity of both beams is measured and compared.

The measurement is performed with the sample solution, which is filled into the cuvette and the absorbance is measured against the reference/blank solution (solution contains all components except the measured compound). Liquids that do not absorb in the UV-VIS spectrum serve as solvents.

The correctness of the wavelength on the spectrophotometer is checked by measuring a holmium filter or a solution of holmium perchlorate. The correctness of measured absorbance is most often checked by measuring standard solution of potassium dichromate and nickel sulphate.

3.4. Infrared spectroscopy

3.4.1. Principle

Infrared spectroscopy uses the interaction between IR radiation and matter. IR radiation is an electromagnetic wave that can be considered as a wave and a stream of particles (photons) at the same time. Radiation can be characterized by velocity c, wavelength λ, frequency (frequency) ν [Hz], wavelength υ [cm-1] and radiation energy E, between which the following relations apply:

h – Planck constant IR radiation is defined by wavelengths λ = 0.78 - 1000 μm. However, for historical

reasons, wavenumbers are much more commonly used as units in IR spectrometry; IR radiation is then defined by the area υ = 12800 to 5 cm−1 (the limit are not sharp, different authors use different values).

Due to the very wide energy range of photons of the IR spectrum (Figure 19), these particles can cause different energy transitions, when they collide with molecules. Therefore, the IR region is divided into three parts:

Near-Infrared (NIR) 12800 - 4000 cm−1 (0.78 - 2.50 μm)

Mid-Infrared (MIR) 4000 - 200 cm−1 (2.50 - 50 μm)

Far-Infrared (FIR) 200 - 5 cm−1 (50 - 1000 μm)

hchE

1

c

24

Figure 19. Electromagnetic spectrum (Source: http://www.apiste-global.com/fsv/technology_fsv/detail/id=1204)

For the purposes of this textbook the authors will focus only on the mid-IR, method based on absorption or radiation by a substance with a wavenumber of 4000–200 cm-1. The electronic state of the molecule does not change, but there are changes in the vibrational and rotational movements of the molecule. The absorbed quantum of energy increases the internal energy of the molecule due to the vibrations of atoms in the molecules and the rotation of individual parts of the molecule.

Both the presence of individual atomic groups in the molecule and the spatial arrangement of the molecule as a vibrating whole can be read from the infrared spectrum. Even in different substances, the same absorption bands of a given wavenumber (characteristic absorption bands), which lie in the range of 4000-1500 cm-1, always corresponds to a certain spatial and binding arrangement of atoms (functional groups). In the area below 1500–200 cm-1, the so-called skeletal vibrations (vibrations of simple bonds) are manifested. There are no two different compounds that have the same spectrum in this area. This area is called the “thumbprint area” and is characteristic of each substance.

Thus, the main use of infrared spectroscopy lies primarily in determining the identity of compounds and their structural arrangement in the molecule. Less often, infrared spectroscopy is used to determine purity.

3.4.2. Instrumentation

Infrared spectrometers work on the two-beam principle and the measured values are registered in the form of absorption spectrum as the dependence of transmittance [%] on the wavenumber in the region 4000-200 cm-1. A Nernst lamp (a mixture of zirconium oxides and yttrium) is used as a source of infrared radiation. A monochromator (prism or reflection grating) is used to obtain monochromatic radiation. All optics must be made of infrared-transmitting material.

MIR spectrum can be measured as the attenuation of the radiant flux after passing through the sample (transmission measurement) or after reflection of radiation (reflection measurement).

Transmission measurement

Transmission measurement is used to measure gases, liquids, transparencies or solids. Liquid samples must be measured in special cuvettes, which are made of e.g. KBr, NaCl, CaF2, BaF2, ZnSe, CsI, AgCl, AgBr, Ge. It is necessary to consider which solvents will be used for the analysis, since the above-mentioned cuvette materials have different solubilities in different solvents. Of course, material that is not sufficiently resistant to the solvent used cannot be

25

used. A wide variety of solvents are used, most often halogenated solvents being used (eg carbon tetrachloride, chloroform) or carbon disulphide due to the small number of characteristic bands they provide in the spectrum. To measure solid samples, a suspension in paraffin oil or a tablet formed by compressing the sample with KBr is prepared.

Reflection measurement

Reflection measurements (Figure 20) are currently the most widespread in practice. The so-called ATR technique is most often used. In setting up this experiment, a thin layer of the sample is pressed against a crystal most often made of ZnSe, Ge, Si or diamond. Each of the materials has its own spectral and physico-chemical characteristics, and the crystal is chosen according to the needs of a particular analysis. Infrared radiation is reflected in the crystal to the sample, where it occurs with a thin (1 μm) layer of the sample and is reflected back to the crystal, where it refracts and continues to the detector with the optical system. The refraction on the crystal can be multiple or only one.

Figure 20. Reflection measurements

The ATR technique can be used to measure both solid samples and liquids, various

semi-rigid materials respectively. While the transmission technique obtains the IR spectrum of a "average" sample, using the ATR technique we obtain the spectrum of the "actual" sample (a specific surface that is pressed against the crystal). In contrast to the transmission spectra, the ATR technique emphasizes the intensity of the bands in the region of low wavelengths compared to the bands at higher wavelengths. Therefore, the spectra obtained by the ATR technique are often mathematically corrected to approximate the transmission spectra.

3.5. Thin layer chromatography

3.5.1. Principle

Thin-layer chromatography is a separation method in which the stationary phase consists of a suitable material applied in a uniform thin layer to the plate (substrate). The separation is based on the principle of adsorption, partitioning, ion-exchange chromatography, or a combination of these mechanisms and occurs by migration (development) of solutes (sample) in a solvent or a suitable mixture of solvents (mobile phase) through a thin layer.

The thin layer of the sorbent may either be a solid phase, in itself involved in the separation process, or may be the carrier of the anchored phase. It is also possible to work

26

with the reverse phase technique (the sorbent is impregnated with hydrophobic solvents). High-performance thin layer chromatography (HPTLC) using smaller uniform diameter silica gel grains is currently being promoted. C1-C∞ modified silica gel (RP-TLC) is also often used. Chiral separation on a thin layer can be realized by impregnating the sorbent with a chiral selector solution.In contrast to paper chromatography, TLC is characterized by simplicity, speed, and ease of evaluation, which has caused its widespread use.

The TLC method is used in every synthetic laboratory. In terms of analytical use, however, it is already receding into background, overshadowed by other modern methods. TLC can be used both to qualitatively determine the presence of a substance (identity), including the separation of isomers or the separation of impurities, as well as for quantitative analysis after elution of the substance from the layer or evaluation of spot area directly on the layer (densitometers, fluorimeters). Reverse TLC can be used to experimentally determine the lipophilicity of substance.

3.5.2. Instrumentation

Glass, aluminium or plastic plates are used as a thin base on which the stationary phase is applied. The stationary phase (sorbent) is mostly formed of silica gel (e.g. Silufol®) or its modification, alumina (e.g. Alufol®) or powdered cellulose (Lucefol®). The layer is strengthened with a binder.

At present, the thin films are manufactured and supplied commercially, e.g. Silufol® UV 254 or 366 which also contains a fluorescent indicator in the sorbent layer enabling detection at the stated wavelengths. To determine the position of the spots of individual substances, it is also possible to use chemical detection by spraying with the reagent to form a coloured product. There is also the possibility of detection using a hot-air gun.

In classical TLC, organic solvents or their mixtures serve as the mobile phase. Chromatography is performed in a sealed chamber which is saturated with vapours of the mobile phase. The most common procedure is the vertical development method. After development and drying of the plate, two-dimensional chromatography can be applied. At the end of development, the position of the mobile phase front is marked and the plate is placed under UV lamp.

3.6. High performance liquid chromatography

3.6.1. Principle

High-performance liquid chromatography is based on the difference in the distribution of substances between two immiscible phases. The mobile phase is the liquid which passes through the stationary phase packed in the column. This method is based on the mechanism of absorption, partitioning, ion-exchange chromatography, or stereochemical interactions.

The main output is the signal from the detector, which is written to the chromatogram, a graphical record of the detector response, the concentration of an eluted substance or other quantity used as a measure of this concentration as a function of time, volume or distance. Ideally, the chromatogram represents a series of isolated symmetrical peaks distributed at baseline. This chromatogram is used for qualitative and quantitative evaluation.

The retention time tR, the time from injection to the maximum of a given peak, is important for the identification of the separated components of the mixture. The dead

27

retention time tM is the retention time of a substance that is not retained in the column at all (it moves through the column at the same speed as the mobile phase). The identification of a given component is based on comparing the retention time of this component and the standard. The method of adding the standard to the separated mixture and performing a second separation is often used. The peak of the identified substance increases with the addition of the standard.

The quantitative representation of the component is determined as the area under the peak. Each chromatograph is equipped with software that is able to integrate the peaks and pinpoint the area under the peak. The content of the substance in the sample is always determined relative to the area of the standard whose exact concentration is known.

Methods:

Method of an external standard – the standard is chromatographed under the same

conditions, but in a different injection

Method of an internal standard – the standard is added directly to the analysed

sample and chromatographed simultaneously

The used chromatographic condition must ensure sufficient separation of the internal standard peak from the peaks of other components of the mixture (the chromatographic method must be sufficiently selective).

High-performance liquid chromatography, especially its modification using the reverse phase (RP-HPLC) has become the most widely used technique, as it allows for the analysis of samples from inorganic ions to polymeric compounds or complex mixtures of natural substances. It also allows you to work with thermolabile, non-volatile, or chiral substances. It can be used to identify substances as well as to determine the content and check their purity. Preparative HPLC can be used to isolate substances.

Reverse HPLC on octadecylsilyl (C18) columns is often used to experimentally determine the lipophilicity of compounds, a parameter that significantly affect the properties of all biologically active compounds.

3.6.2. Instrumentation

The apparatus consists of a pumping system, a dosing device, a chromatographic column including a thermostat, a detector, and a data processing system/software. The mobile phase is pumped to the system from one or more reservoirs and usually flows at a constant rate through the column and then through the detector.

The separation process can be performed:

Isocratic – a constant composition of the mobile phase is used during separation

Gradient – during the separation, the composition of the mobile phase changes (to

one part of the mobile phase an increasing amount of the other mobile phase is mixed)

Principle of substance separation:

The dispenser injects the sample solution into the mobile phase stream, which is carried to a chromatographic column located in a thermostat. The substances are carried by the mobile phase through the column through a stationary phase, on which the sample is separated into individual components. From the column, the individual separated

28

components enter the detector, which provides (registers) a response (signal) corresponding to changes in the concentration of substances in the mobile phase leaving the column.

Device

The mobile phases are pumped by a high-pressure pump, which must allow a constant pulseless flow of the mobile phase at a low speed (0,1-10 ml/min) at high pressure (up to 40 MPa). Pumping systems can be equipped with a degasser – a device for removing dissolved air in mobile phases. Microprocessor-controlled systems are able to accurately deliver a mobile phase either with the constant composition (isocratic) or a composition that varies according to a predetermined program (gradient). This mobile phase is mixed from multiple reservoirs.

The dosing device dispenses the sample solution into the flowing mobile phase to the upper end of the column. It is able to work at a high pressure. Constant or variable volume dosing loop devices for manual or automatic dosing are used.

The detector converts the results of the separation in the column into a registrable form. The general requirements are:

Versatility

Sensitivity (concentration ng to µg/ml)

Reproducibility and linearity of response

Independence of the response to changes in the compositions of the mobile phase

UV-VIS spectrophotometers (with a selectable wavelength (190-800 nm) are most often used), including diode array detectors (they scan the entire absorption spectrum several times per second, resulting in a 3D chromatogram as the dependence of absorbance on wavelength over time). According to the properties of substances and the purpose of the analysis, fluorescence spectrophotometers as well as refractometric, fluorometric, mass and electrochemical detectors can be used.

The signal from the detector is processed by the chromatographic software to obtain a chromatographic record (chromatogram) in the form of peaks for a qualitative and quantitative evaluation of the analysis.

Mobile phases

Less polar solvents are used in normal phase chromatography. To achieve reproducible results, it is necessary to control the amount of water in the mobile phase. In reverse phase chromatography, aqueous mobile phases are used, or phases with a suitably adjusted pH, mixed with or without an organic solvent. The mobile phase components are filtered to remove particles larger than 0,45 µm. Multicomponent mobile phases are usually prepared by measuring the required volumes and then mixing them due to volume concentration. All components of the mobile phase must be of suitable quality, organic solvents must not contain stabilizers and other additives to be permeable to wavelengths when detected in the UV range. Columns and stationary phases

The HPLC columns are steel tubes 2-25 cm long with an inner diameter of 2-8 mm filled with the stationary phase. The column packing must be homogenous and uniform, therefore columns filled and tested by the manufacturer are used. Stationary phases with a particle size of 1,5 to 10 µm are used for analytical separations (the more regular the shape of the

29

particles, the higher efficiency of the column). An interesting type of columns are the so-called monolithic columns, which are made of highly purified polymeric silica gel or its C8, C18 modifications. The connections between the column, the detector, and the dispenser are made of steel capillaries with an inner diameter of 0,5 mm.

Many types of stationary phases are used in liquid chromatography, the quality of which (size, shape, porosity, uniformity, and structure) plays a major role in the quality of separation. The following can be used for HPLC separation:

Silica gel, alumina, porous graphite, zirconium – used in separation with normal

phases; the principle of adsorption chromatography

Resins, polymers with acidic or basic groups; the principle of ion-exchange

chromatography

Porous silica gel or polymers; the principle of separation based on differences in the

volumes of molecules

Chemically modified silica gel, porous graphite, zirconium and synthetic polymers –

used for reverse-phase separation (RP); the principle of separation consists in the

combination of interactions of the analyte with the mobile phase and the mobile

phase with the stationary phase and on partition chromatography

Special chemically modified stationary phases for the separation of enantiomers; the

principle of separation based on the theory of the so-called three-point interaction

consisting in a combination of hydrogen interactions and hydrophobic bonds, charge

transfer and/or electrostatic, specifically dipole-dipole attractive and repulsive forces

In RP-HPLC columns, the liquid stationary phase anchored on a suitable support is not used, because the mechanically anchored liquid phase is not stable at high pressure in the columns. The above-mentioned chemically modified stationary phases are used. The most common modifications of the hydroxyl groups of silica gel are:

octadecyl = Si-(CH2)17-CH3

octyl = Si-(CH2)7-CH3

phenyl = Si-(CH2)n-C6H5

cyanopropyl = Si-(CH2)3-CN

aminopropyl = Si-(CH2)3-NH2

amidopropyl = Si-(CH2)n-CONH2

diol = Si-(CH2)3-OCH(OH)-CH2-OH

Silica gel columns can be heated to a maximum of 60 °C. Unless otherwise stated by the manufacturer, silica gel-based reverse stationary phases columns are stable in mobile phases at pH 2-8.

For these mentioned limitations, zirconia, titanium dioxide or variously modified organic polymers are used as carriers for the chemically bonded phase. Expansion of the zirconium columns has been observed, in which similarly to silica gel-based columns, the stationary phase of zirconia is chemically modified with various groups. Depending on the stationary phase base used and its subsequent modification, these types of columns may be stable in the pH 1-14 pH and up to 100 °C.

Optically active substances need to be separated more and more often. Separation can be performed in two basic ways:

30

1. using a non-chiral stationary phase and a chiral mobile phase (a chiral selector is added

to the mobile phase)

2. using a chiral stationary phase (chiral selector is attached to inert support) and a non-

chiral mobile phase

Chiral stationary phase columns are more commonly used. Silica gel is most often used as an inert carrier. Inert carriers can be modified with natural or synthetic selectors. The most used are:

polysaccharides – derivates of cellulose, amylose, starch, and cyclodextrins

proteins – albumin, orosomucoid, avidin

macrocyclic antibiotics – vancomycin, avoparcin, teicoplanin or ristocetin A

macrocyclic polyethers so-called crown ethers (synthetic selectors)

3.7. NMR spectroscopy

3.7.1 General information

NMR spectroscopy (nuclear magnetic resonance) is a method in which the energy state of nuclear spins changes in a strong magnetic field after the absorption of radiofrequency radiation.

The basic building blocks of the nucleus of atoms (protons and neutrons) rotate around their own axis and thus have an angular momentum p, called spin. Protons and neutrons have a spin quantum number I = ½. In isotope nuclei with an even number of both protons and neutrons, the spins of the particles are paired, so that the resulting spin of the nucleus is I=0. Such nuclei (e.g. 12C, 16O) have a zero spin and do not provide NMR signals. Nuclei with an odd number of protons, neutrons or both types of particles do not have spins paired and their I>0. Because the movement of electrically charged particles along a closed path is associated with the formation of a magnetic field, nuclei with I>0 (e.g. 1H, 13C, 17O) have their own magnetic moment:

𝜇 = 𝛾𝑝 γ – gyromagnetic ratio, characterizing each isotope If we place a sample containing an isotope with a non-zero magnetic moment in a strong magnetic field with induction of the magnetic field B0, splitting the energy levels of its nuclear spin occurs, see figure 21. The potential energy of the nucleus E is given by the relation:

𝐸 = −𝜇𝐵0 And for the component of the magnetic moment in the direction of the field B0:

𝜇 = 𝑚ℎ𝛾

2𝜋 m – magnetic quantum number

h – Planck constant

The magnetic quantum number takes the values m= I, I-1, I+1, -I. The number of generated energy levels is determined by the value of the spin quantum number I (number of states =2I +1), therefore for isotopes with I= ½, such as 1H and 13C, nuclear spins occupy two energy states with magnetic quantum number m= + ½ and m= - ½. Their energy can be calculated from the relation:

31

𝐸+

1

2

= −1

2(

ℎ𝐵0𝛾

2𝜋) 𝐸

−1

2

= +1

2(

ℎ𝐵0𝛾

2𝜋)

Figure 21. Nuclear magnetization M0 at equilibrium. Magnetic moments directed in accordance with the external magnetic field B0 belong to spins in the + ½ state, oppositely orientated belong to spins in the – ½ state.

According to the laws of quantum mechanics, each spin can only be permanent at one

energy level, so it can have only one of two possible orientations. Transitions between these levels can be caused by radiation whose frequency satisfies the condition:

∆𝐸 = 𝐸−

1

2

− 𝐸+

1

2

= ℎ𝑣 E – energy difference between levels

Using the previous relationships, it is then possible to write:

𝑣0 = ∆𝐸

ℎ=

𝛾𝐵02𝜋

This relationship is known as the resonance condition, frequency v0 is the so-called Larmor frequency. However, it is often given in the form:

𝜔 = 𝛾𝐵0 - angular velocity Because the differences in ΔE levels are small, only radiofrequency energy (frequency

in MHz) is sufficient to excite these molecules. At the energetically lower level there are nuclei whose projection of the nuclear

magnetic moment µ is oriented in accordance with the external magnetic field B0. This orientation corresponds to the magnetic quantum number m= + ½. The nuclei at higher energy levels have the resulting magnetic moment oriented against the direction of the magnetic field m = - ½, see Figure. 21.

32

Note: At equilibrium at temperature T = 300 K and magnetic field induction B = 1 T, according to Boltzmann´s distribution law, the ratio of the number of proton spins at an energy level is:

𝑛+

1

2

(𝑚 = +1

2) and 𝑛

−1

2

(𝑚 = −1

2), therefore:

𝑛+

12

𝑛−

12

= exp (∆𝐸

𝑘𝑇) = 1. 000006 k – Boltzmann´s constant

The number of nuclei at a lower energy level is therefore only slightly larger than at a higher energy level. The result of this surplus is the vector sum of all nuclear spin magnetic moments – nuclear magnetization M0.

When measuring NMR spectra using pulse methods, a suitable radiofrequency pulse balances the number of both energy levels and the magnetization M0 deviates from its equilibration position (from the direction of the axis of the external magnetic field B0). Because magnetization is associated with angular moment of, it does not return to the direction of the magnetic field immediately after the end of the pulse, but behaves similarly to a flywheel mounted outside the centre of gravity, whose axis of rotation is inclined to the gravitational field. The result is a precession in which magnetization maintains its magnitude and inclination to the magnetic field, and rotates around the direction of the magnetic field B0 with Larmor frequency.

In addition, the relaxation processes gradually restore the disturbed equilibrium state. After a certain time, characterized by the time of the so-called spin-lattice relaxation T1, the equilibrium distribution of spins at both energy levels stabilizes again. This restores the magnetization component in the field direction. On the contrary, the magnetization component perpendicular to the direction of the magnetic field decreases down to zero value with the time of so-called spin-spin relaxation T2.

Just as a rotating magnet excites alternating voltage in the alternator stator coils, so also a rotating magnetization vector excites alternating voltage in the receiver coil, which is wound around the sample in the NMR spectrometer. The time course of the voltage (signal) induced by nuclear magnetization in the coil is commonly referred to as FID (Free induction decay), it is indicated in figure 22. and can be thought of as a fading tone when you press the piano key.

Figure 22. FID – dependence of signal s on time t for: one (a), two types (b)of nuclei, to which answer one or two signals

33

If the sample contains only one type of atom of one isotope as e.g. CHCl3 (Figure 22.a), the decrease in the amplitude of damped oscillations is exponential and is characterized by the spin-spin relaxation T2. Frequency of oscillations is the Larmor frequency. In the case of a more complex sample, the resulting FID is an interferogram or superposition of FIDs originating from different nuclei of the same isotope (Figure. 22.b). In order to obtain the resonant frequencies of individual types of nuclei and the intensity of their signal response, it is first necessary to convert the FID, i.e. the time dependence of the signal on the dependence of the signal on frequency, or on the NMR spectrum. This conversion is performed on a computer by the so-called Fourier transform. The NMR spectrum then contains information on the structure of the substance, which is stored in three parameters:

1. Position of signals (chemical shift)

2. Integral signal strength

3. Multiplicity (fine structure) of signals

3.7.2 1H NMR spectroscopy

Signal position – chemical shift

Relationships between frequency of absorbed radiation and magnetic induction B0 is

expressed in the resonant condition:

𝑣0 = ∆𝐸

ℎ=

𝛾𝐵02𝜋

Under this condition, the resonant frequency should be constant for all nuclei of the same

isotope. However, in a molecule, individual nuclei are bound into groups and are thus affected

by different electron environments. The electron environments changes („shields”) the

magnetic field at the nucleus, so in the place of the nucleus applies the magnetic induction

Blok relation:

𝐵𝑙𝑜𝑘 = 𝐵0(1 − 𝜎) σ – shielding constant

Determining the shielding constant is experimentally demanding, and therefore the

relative values of resonant frequency relative to the standard frequency are given in practice.

Tetramethylsilane (TMS) is usually used as a standard, which is added directly to the

measured sample as a so-called internal standard. The distance of the resonant signals from

the TMS signal is measured in Hz. Since the value depends on B0, the chemical shift [ppm]

was defined as a quantity that is independent of magnetic field induction:

𝛿 = 𝑣 − 𝑣𝑠𝑡

𝑣0 106

v – frequency of measured nucleus vst – frequency of measured standard v0 – working frequency of spectrometer for given isotope

Chemical shift is expressed in ppm (parts per million). The value of the chemical shifts TMS is

according to the relation:

𝐸+

1

2

= −1

2(

ℎ𝐵0𝛾

2𝜋) 𝐸

−1

2

= +1

2(

ℎ𝐵0𝛾

2𝜋)

34

equal to 0 ppm. The range of chemical shifts in hydrogen 1H NMR spectra is most often

0-20 ppm.

Table 1 shows the chemical shifts of the protons, according to which the proton signal

can be assigned to a certain structural grouping. If protons are bound in the same way, and

thus have the same „environment”, they have the same resonant frequency. Such nuclei are

called magnetically equivalent (but not vice versa). For example, in benzene or cyclohexane,

due to symmetry, all protons are equivalent and provide only one signal in the 1H NMR

spectrum. Similarly, all three protons in the -CH3 group are equivalent due to the free rotation

around a single bond, and in the spectrum, they will correspond to only one signal. Also

compounds Si(CH3)4 and symmetrically substituted ethane X-CH2-CH2-X (both free rotation

around a single bond and symmetry of the molecule apply here) give only a single signal in

the 1H NMR spectrum, corresponding to twelve in the case of tetramethylsilane and four

equivalent protons in the case of ethane.

The chemical shift is affected by a number of factors (electronegativity of substituents,

magnetic anisotropy, steric effects, temperature, concentration, solvent, etc.). The most

important of these is the electronegativity of substituents. With the increasing

electronegativity of neighbouring atoms or groups, the shielding decreases and the chemical

shift value increases. Temperature, concentration and solvent affect most significantly the

value of the chemical shift of protons bound to heteroatoms (-OH, -NH, -SH).

Figure 23 is a 1H NMR spectrum of propylparaben. There are six signals in the

spectrum, corresponding to the six groups of protons contained in the propylparaben, i.e.

-CH3, -CH2-, -CH- and HO-. They differ with their chemical shifts, which are in the accordance

with the values given in tab. 1. The signal with the chemical shift of 7,27 ppm, which is also

found in the spectrum, corresponds to the residual signal CHCl3 in CDCl3, which was used as

solvent in this case.