Work with solutions

Measuring vessels and other tools in the chemical laboratory
When preparing solutions and handling liquids, we use various laboratory containers and tools. They differ in the purpose for which they are to be used and in accuracy. Maintaining the temparature is important because the density of the liquid changes with temperature.

Measuring vessels are usually calibrated 20 °C (in the US 25 °C). The calibration temperature is marked on each measuring container.


 * {| class = wikitable

|+ Aids for measuring the volume of liquids ! Aid                               !! Usual volume range !! Accuracy | | Erlenmeyer flask, beaker   || 5–5000 ml                  || indicative only | | Measurign flask                      || 5–2000 ml                  || high | | Graduated cylinder                       || 5–2000 ml                  || medium | | Burette                                  || 1–100 ml                    || high | | Pasteur pipette, dropper      || 1–5 ml                       || small | | Glass pipette                     || 1–100 ml                    || high | | Automtic pipette                 || 5–5000 &mu;l             || high | | Automatic dispenser            || 0,1–100 ml                 || medium | | Micro syringe                        || 0,5–1000 &mu;l          || high | | Piston valve dispenser            ||1–500 ml                    || medium
 * }

Beakers


Beakers are used for indicative determination of liquid volumes. In addition to rough measuring of volumes beakers are mainly used for dissolving substances, diluting liquids, heating and other laboratory operations. Because of the low measurement accuracy, they are usually not even classified as measuring containers.

Measuring flasks and measuring cylinders
Measuring flasks and measuring cylinders are calibrated "for filling", which is marked on them with the mark D according to the Czech "dolít" or IN according to the English "include". After filling up to the appropriate line, the liquid inside the container has the exactly indicated volume. If we pour the liquid into another container, a certain amount will remain in the form of a thin film or drops on the walls, so by pouring we transfer less than the indicated volume.

Volume is usually given in milliliters. When measuring, the container must stand on a firm, horizontal support. The correct volume is measured if the meniscus of the liquid touches the mark on the container with its lower edge. Measuring cylinders are only used for approximate measuring, measuring flasks are used to prepare solutions with a precise concentration.

Burettes, pipettes, dispensers and syringes
Burettes, pipettes, dispensers and syrines measure the volume of liquid taken into antoher container.

Pipettes and burettes are usually calibrated „for pouring“, marked V according to Czech "vylít" or Ex according to the English "exclude". The liquid flowing from the respective lines has the indicated volume. We do not blow out the contents of the pipette, even if a drop remains in the tip. Its volume is taken into account during calibration.


 * Burettes

They are used in titrations, or where the same volume of liquid is repeatedly measured. These are glass or plastic calibrated tubes closed by a tap. The burette is fixed vertically to the stand using a holder. With the tap closed, it is carefully filled with the appropriate liquid using a funnel. The funnel is removed and by slightly opening the tap, such an amount of liquid is released that its lower meniscus touches the mark. Then the burette is ready for titration. The titration agent is discharged through the tap and its volume is monitored on the scale. One of the most important actions when working with a burette is the correct reading of the volume. It is always read twice on the burette. The first time when determining the zero mark, the second time when subtracting the drained volume. Since the change in volume is subtracted, the method of subtraction does not matter much. However, it must always be subtracted in the same way.


 * Automatic burettes are used in routine laboratories.


 * Glass pipettes


 * They are rarely used to measure volumes in modern routine laboratories and are being replaced by semi-automatic dispensers. The volume of glass pipettes can be different, from 1 to 100 ml. They can be either undivided, designed to measure a single volume, or divided - usually in milliliters and tenths of milliliters. The scale can point from the tip towards the top edge or vice versa.
 * For safety reasons, we never suck the solution into the pipette by mouth - different types of attachments or pistons are used to draw it.


 * When suctioning, the pipette must not rest on the bottom of the container. Before measuring the sample, the pipette is first filled with the solution and the collected volume is drained into the waste container. Only then is the exact volume taken and transferred to a container for further processing. The solution must never enter the pipette tip.




 * Automatic pipettes (pipettors, micropipettes, microdispensers)
 * One option for measuring small volumes is the use of automatic pipettes.


 * Microsyringes
 * They are used for precise dosing of small amounts (0,1–1000 &mu;l) of liquids. They consist of a needle attached to a graduated glass cylinder in which a piston moves. Individual types differ in needle and piston diameters.




 * Piston valve dispensers

thumb|100px|Pístoventilový dávkovač
 * They consist of a piston with a scale, which is mounted on storage bottle. They enable the repeated dosing of a certain volume of liquid from the storage bottle. Dosers intended for dosing of aggressive chemicals (e.g. strond acids) have glass parts made of borosilicate glass, plastic parts that are in contact with the liquid are made of PTFE, other parts are made of PE or PP. In modern types of dispensers, dosing is automated using control modules.

Automatic pipettes
According to the control method, we distinguish between manual and electronic automatic pipettes. In manual pipettes, the plunger is moved with the thumb using the control button. The correctness and accuracy of pipetting is significantly influenced by the experience and skill of the worker. In the case of electronic pipettes, the piston is moved by an electric motor. Compared to manual methods, it also offers programming of the pipetting method. Depending on the nature of the liquid. you can choose a different speed of piston movement when sucking in an expelling the liquid.

A disposable tip is attached to the body of the pipette (also called pipettor). The pipetted liquid comes into contact only with this tip.

According to the principle of their operation, automatic pipettes can be divided into two basic types:
 * „Air displacement“ pipettes
 * This type of pipette uses the so-called air cushion principle. A certain volume of air always remains between the piston and the liquid. The volume of liquid drawn into the tip by the pipettor may differ slightly from the volume of air drawn in or pushed out by the piston, depending on the density and viscosity of the pipetted liquid, the wettability of the tip surface by the pipetted liquid, temperature and atmospheric pressure, and other influences. Therefore, the pipettor must be regurarly calibrated and adjusted.


 * Pipettes of this type are distinguished according to their design as single-channel (intended for pipetting one volume of a given liquid in time) or as multi-channel (most often eight or twelve-channel) intended for simultaneous pipetting of the same volume of a given liquid into several wells in a microtiter plate. Each channel in multi-channel pipettes has its own piston, therefore it is not necessary to use all channels at one (less than 8 or 12 tips can be connected).


 * Automatic pipettes are designed either for one fixed volume or are adjustable for multiple volumes. Changing the volume setting is possible within a certain range (e.g. 10-100 &mu;l) using the adjusting screw or knob.


 * „Positive displacement“ pipettors
 * This type of pipette sucks liquid into the tip directly without creating an air cushio, i.e. the piston is in direct contact with the measured liquid. The liquid sucked into the tip (without air bubbles) is discharged all at once (syringe type) or in steps of the same volume (stepper pipette). This type of pipettor is convenient to use for highly viscous or volatile liquids, or for repetitive pipetting.

Direct pipetting
This is the most commonly used pipetting technique. During direct pipetting, a precisely set volume is sucked into the tip and in the next step it is completely pushed out of the tip into the selected container. Because a certain amount of liquid remains on the inner surface of the tip as a thin film, it is necessary to wet the tip with the measured liquid before pipetting. The direct pipetting technique is used for measuring most aqueous solutions, buffers, dilute acids and bases.



Method

 * 1) Place the tip on the dispenser. Press the controller button to the first position (a small resistance must be overcome when pressing the button).
 * 2) Dip the tip of the dispenser about 2-3 mm below the solution level. Slowly release the pressed button on the controller while sucking the sample into the dip.
 * By slowly sucking the liquid into the tip, the possible formation of turbulence is limited, which can cause the formation of aerosol and gas bubbles coming out of the liquid. The optimal speed depends on the properties of the liquid (its density, vapor tension and viscosity). center|250px
 * 1) * Always check whether air bubbles have entered the tip (e.g. when the piston is opened more sharply or the tip is incorrectly fitted). center|40px
 * 2) * For greater pipetting accuracy, remove your thumb completely from the controller button once it reaches the home position.
 * 3) Slowly withdraw the tip from the liquid. Some of the contents of the tip may be lost when pulled out quickly. Wait, especially for larger 500–5000 &mu;l pipettors, about 1-3 seconds before pulling the tip ouf of the liquid.
 * 4) When expelling the liquid, hold the tip at a slight angle agains the wall of the container (10–45º), just above the solution already in it, and smoothly press the control button with your thumb to the first positio. Wait about 1 second and continue to quickly press the controller button to the second position (you will feel more resistance when pressed). Make sure that no droplets of liquid remain in the tip or splash on the wall of the container. center|250px
 * 5) Hold down the controller button and pull the tip out along the wall of the container. Now enable the controller button.

In direct pipetting, a certain error is created by the fact that a very thin film of the tranferred liquid remains on the inner surface of the tip. With the mentioned procedure, we measure a slightly smaller volume than is set on the pipette, while the error depends mainly on the properties of the pipetted liquid and the material from which the tip is made. This error can be eliminated by wetting the inner surface of the tip with the measured liquid before pipetting. In practice, this means that we first suck the solution into the tip using the procedure described above, but instead of measuring it into the target container, we return it back to the storage container. At this moment, a film of pipetted liquid is formed on the inner wall of the tip, in the case of colorless solutions it is invisible to the eye with the correct technique. This is followed by measuring the liquid exactly according to the above mentioned procedure (only we do not insert a new tip.) Since the amount of liquid that remains in the tip is practically constant, we now measure the actual set volume.

If drops remain on the outer wall of the tip, it is possible to wipe them a cotton wool with a light movement from top to bottom. Never touch the mouth of the tip with the cotton wool, otherwise you will suck out some of the liquid inside. center|250px

Reverse pipetting
In reverse pipetting, we draw a larger volume of liquid into the tip than we want to measure, and in the next step we push out the volume set on the pipette from the tip. This method of pipetting gives better results when working with viscous or highly volatile liquid, strongly wetting liquids and solutions that foam. It is also suitable for measuring very small volumes. After pipetting, there is always a residue of liquid in the tip, which can be squeezed back into the storage container or into the waste before removing the tip itself.

Postup

 * 1) Stiskněte tlačítko až do druhé polohy (pocítíte nejdříve slabý a poté větší odpor pístu při stisku tlačítka ovladače).
 * 2) Ponořte špičku pipety asi 2–5 mm pod hladinu roztoku. Pomalu povolujte píst za současného nasátí vzorku do špičky.
 * 3) Pomalu vytáhněte špičku z kapaliny a odstraňte kapky ulpělé na vnější stěně špičky dotekem špičky o okraj nádoby.
 * 4) Při vytlačování daného objemu kapaliny držte špičku v mírném úhlu proti stěně nádoby těsně nad roztokem, který v ní již je, a pomalu plynule stiskněte palcem tlačítko ovladače do první polohy.
 * 5) Držte tlačítko ovladače zmáčknuté v této poloze a vytáhněte špičku z nádoby ven.
 * 6) Část kapaliny, která zůstane ve špičce, vytlačte stiskem tlačítka ovladače do druhé polohy zpět do původní nádoby nebo do odpadu.
 * 7) Podržte tlačítko ovladače zmáčknuté a vytáhněte špičku z kapaliny ven a pak povolte tlačítko ovladače.

Opakované pipetování
thumb| upright=1.5 |Schema pipetování-opakující se Tento způsob pipetování je určen pro opakované pipetování stejného objemu, např. pro přidávání činidla do série zkumavek nebo do jamek v mikrotitrační destičce. Jedná se vlastně o opakující se reverzní pipetování. Po nasátí kapaliny do špičky se opakují kroky 2 až 4.

Pipetování heterogenních vzorků
Technika vhodná pro pipetování heterogenních vzorků jako je krev, kdy není snadný proplach špičky před pipetováním. Podobá se přímé technice, ale špička se předem nesmáčí odměřovanou kapalinou. Namísto toho se po přenesení kapaliny opakovaně propláchne roztokem, s nímž se odměřovaná kapalina mísí. thumb | upright=1.5 | Schema pipetování-heterogenních vzorků

Postup

 * 1) Stiskněte tlačítko do první polohy a ponořte špičku dávkovače asi 2–5 mm pod hladinu roztoku.
 * 2) Pomalu povolujte píst za současného nasávání vzorku do špičky.
 * 3) Pomalu vytáhněte špičku z kapaliny a odstraňte kapky roztoku ulpělé na vnější stěně špičky vytažením špičky podél stěny nádoby.
 * 4) Ponořte špičku dávkovače do cílového roztoku.
 * 5) Stiskněte ovládací tlačítko do první polohy a pak ho pomalu povolte do původní polohy. Tím dojde k nasátí roztoku do špičky. Špičku nevyndávejte z roztoku a opakujte tento krok, dokud vnitřní stěna špičky není čistá.
 * 6) Po stěně povytáhněte špičku nad hladinu roztoku a vyprázdněte ji stiskem tlačítka ovladače do druhé polohy.
 * 7) Podržte tlačítko ovladače zmáčknuté a vytáhněte špičku z nádoby podél stěny ven a pak povolte tlačítko ovladače.

Příprava roztoků o dané koncentraci
Hmotnostní koncentrace: &rho;B = mB / V (g/l)

Látková koncentrace (molarita):		cB = nB / V	(mol/l)

Hmotnostní zlomek:			wB = mB / m

Zřeďování a směšování roztoků:		c1V1 + c2V2 = c3(V1 + V2)

Číslo zředění (zřeďovací faktor): D = (Vkonečný / Vpůvodní)

D-krát zředěný roztok o objemu Vkonečný připravíme z 1 dílu původního roztoku (Vpůvodní) a (D−1) dílů rozpouštědla (např. 5krát zředěný roztok se získá smícháním 1 dílu původního roztoku a 4 dílů vody).

V anglosaské terminologii se setkáváme též s pojmem zřeďovací poměr, který vyjadřuje 1 díl původního roztoku ku D dílům celkového objemu (1/D = Vpůvodní / Vkonečný). Podle tohoto pojetí roztok zředěný 1: D je totéž co D-krát zředěný roztok (např. roztok zředěný 1: 5 je roztok připravený z 1 dílu původního roztoku a 4 dílů vody, tj. 5krát zředěný).

Praktická úloha Příprava roztoků o dané koncentraci

Fotometrické ověření koncentrace
Při průchodu elektromagnetického záření z oblasti ultrafialové nebo viditelné části spektra měřeným roztokem dochází k jeho absorpci. Velikost absorpce závisí na vlnové délce záření, na koncentraci absorbující látky v roztoku a na tloušťce měřené vrstvy. Při dané vlnové délce záření existuje mezi koncentrací absorbující látky a veličinou nazývanou absorbance (A) přímá úměra. Tuto závislost vyjadřuje Lambertův-Beerův zákon: A = &epsilon;&alpha;c l, kde&epsilon;&alpha;je molární absorpční koeficient (jeho hodnota odpovídá absorbanci látky o koncentraci 1 mol/l a tloušťce měřené vrstvy 1 cm), c je látková koncentrace (mol/l) a l tloušťka měřené vrstvy (cm). Daný vztah platí pouze pro monochromatické záření a pro zředěné homogenní roztoky. Pro měření absorbance se zpravidla volí vlnová délka odpovídající absorpčnímu maximu stanovované látky.

Praktická úloha Fotometrické ověření koncentrace