Laboratory Exercises in Medical Chemistry III

Transcription

1 Laboratory Exercises in Medical Chemistry III 1 st year, General Medicine Faculty of Medicine in Pilsen Charles University Name: Confirmation of the attendance at the labs Study group: Date: (stamp, tutor's signature) Date of report delivery: Report accepted returned for revision

2 Programme 3 working places (groups of exercises) 45 minutes per each working place a) Colorimetric determination of ph - determination of ph using universal indicator strip (wide range strip) - determination of ph using "MULTIPHAN" indicator strip (narrow range strip) - determination of ph by comparison with set of ph standards (buffers) b) Demonstration of the function of buffers - demonstration of resistance to change in ph in buffered solutions (compared to unbuffered) c) The buffer effect of acetate buffer - estimation of the ph where the acetate buffer exhibits the best buffering properties A lab coat is required to be worn over your clothes, when working in a lab! After the work is done, clean your working place so that it is the same as it was in the beginning! Before you leave the lab, ask laboratory assistant for a stamp and your tutor for signature!

3 a) Colorimetric determination of ph The ph indicators are substances that change colour according to the ph of the solution into which they are introduced. They are weak acids or bases whose conjugate forms differ in colour. The following equilibrium exists in solution: HI H + + I - The proportion of HI to I - present is thus determined by the ph, and the Henderson-Hasselbalch`s equation for the system is: [ I - ] ph = pk i + log [HI] The species HI and I - have different colour, i.e. absorb light of different wavelengths, because of differences in their molecular structure. The changes in ph are accompanied by changes in [I - ] / [HI] ratio, and, therefore, by changes in colour. Human eye can register 10% change in [I - ] / [HI] ratio and this way the ph can be estimated. It is obvious that the colour changes occur within the range ph = pk i ± 1. methyl red ph = pki ± 1 Objective: Determine the ph of the sample There is one test tube marked with number of your sample in a rack on your working place. This test tube contains exactly 10.0 of the unknown sample. Your task is to determine ph of this sample by 3 different methods (universal indicator strip, "MULTIPHAN" indicator strip, comparison with a set of ph standards). No new sample available at all! Sample number: The universal ph indicator strips usually cover the full 0 14 ph range, but have low sensitivity of 1 ph unit. Intermediate range strips provide a sensitivity of ph units and narrow range strips provide a sensitivity of ph units. Intermediate and narrow range strips are often manufactured for special clearly defined use, e.g. urine ph measurement. Urine ph is somewhere between 4.5 and 7.5 therefore indicator strip for such purpose may be limited in range by these values. 1) universal indicator strip wide ph range: 0 12 limited accuracy (accurate to ± 0.5 ph unit) To use universal indicator strip test paper is by far the simplest way to measure the ph. Take a piece universal indicator strip with tweezers and immerse it briefly (for approx. 1 second) directly into the test tube with your unknown sample to be tested. Be careful to prevent fall of indicator strip deep into the test tube. If this happens, remove the strip as soon as possible! Paper strip is impregnated with indicators that may bleed into the solution and change its colour. This could impair further experiments with the sample. Immediately compare the colour of the wet indicator paper with the ph colour chart printed on the box and read the ph value. ph determined by universal indicator strip: 1

4 2) "MULTIPHAN" indicator strip ph range: "universal" indicator segment segments for comparison narrow ph range of individual strip better accuracy (accurate to ± ph unit) The kit consists of several indicator strips, each of them for different narrow ph range. First you have to select an appropriate strip so that ph you expect to measure is within the range. You already know approximate ph of your sample (ph determined by universal indicator strip). ph range of selected indicator strip: Take this indicator strip with a pinzette and immerse it briefly (for approx. 1 second) directly into the test tube with your unknown sample to be tested so that all the fields come into contact with the solution. Compare the colour of the indicator zone in the middle with the coloured segments placed on both sides. Look for the best match. Read the ph value from the enclosed schematic table. From the schematic table enclosed in the kit, copy the corresponding ph values into the fields.??? MIDDLE indicator zone?? Mark the zone which colour is the most similar to the colour? of the middle indicator zone ph determined by "MULTIPHAN" indicator strip: 2

5 3) by comparison of the colour of acid base indicator in set of ph standards (buffers) Prepare the set of 8 buffers according to the table: Solution of acetic acid (CH 3 COOH) and solution of sodium acetate (CH 3 COONa) are located in automatic burretes. These solutions are shared with a student working on place "b) The buffer effect of acetate buffer". If the burretes are being used, please skip to "Demonstration of the function of buffers" and return to this exercise when possible. Automatic burrete This hole must be closed by your finger. Air is pumped inside the reservoir bottle. (Reasonably! Watch what is happening!) When the solution inside the burrete is above zero level, uncover the hole (remove your finger). The level is automatically adjusted to zero. Standard number CH 3 COOH (100 mmol/l) CH 3 COONa (100 mmol/l) ph Use Henderson-Hasselbalch equation to calculate ph in individual test tubes. ph = pk a + log CH3COONa CH3COOH pk a = 4.75 Add exactly 20 drops of the bromocresol green (acid base indicator) into all eight test tubes. bromocresol green: ph interval of colour change: (acidic) yellow blue (alkaline) Add exactly 20 drops of the bromocresol green into the test tube with unknown sample. (The test tube already contains exactly 10.0 of the sample = volume equal to volumes of ph standards prepared) Mix the content of all the test tubes thoroughly. Close the test tube with rubber stopper and slowly turn the test tube upside down. Compare the colour of your unknown sample with the scale of ph standards (8 test tubes prepared). Find the best match. You may use the"comparator" (cube with holes for test tubes) to help you. Colour in the sample test tube is the most similar to standard number: ph determined by this method: 3

6 b) Demonstration of the function of buffers Buffers are solutions used to maintain relatively stable ph. ph changes very little when a small amount of strong acid or base is added. A buffer solution consists of a mixture of a weak acid and its conjugate base (or a weak base and its conjugate acid). Henderson Hasselbalch equation describes the ph of buffer solution: ph = pk a + log buffer base buffer acid where pk a is the negative log of the acid dissociation constant --> ph is given by: pk a of an acid, from which is the buffer derived the ratio of buffer base / buffer acid present in a solution For the demonstration, we will use "phosphate buffer" composed of sodium hydrogen phosphate and sodium dihydrogen phosphate. Use Henderson-Hasselbalch equation to calculate volumes of components needed to prepare 10 of the phosphate buffer with ph = 7.0. pk a = 7.21 What you have: solution of sodium hydrogen phosphate c = 100 mmol/l solution of sodium dihydrogen phosphate c = 100 mmol/l Calculation: Component Formula Volume needed () sodium hydrogen phosphate sodium dihydrogen phosphate There are two titration flasks on your working place. Pipette (using glass pipette and rubber suction bulb) exactly 10.0 ml of WATER into one of the titration flasks. Into the second titration flask, pipette (using glass pipette and rubber suction bulb) exactly the calculated volumes of both components of phosphate buffer. Add 2-3 drops of indicator (methyl red) into both titration flasks. Notice the colour of the solution. Methyl red is a ph indicator with interval of colour change: (acidic) red yellow (alkaline) Fill the burette with standard solution of HCl (c = mol/l). Adjust the level of standard solution in a burette to zero and add HCl (i.e. strong acid) into the solutions in titration flasks until colour change of the indicator (pink red colour). Read the volume of HCl used to reach this point. Volume in titration flask ph Volume of HCl needed to change the ph water phosphate buffer Make conclusions: 4

7 c) The buffer effect of acetate buffer Buffer properties depend mainly on pk and also on the base/acid ratio. The best buffer effect is observed when ph = pk, i.e. when the ratio buffer base/buffer acid = 1. There is a set of 10 titration flasks on your working place. Use a marker to number them from 1 to 10 (if it is not already done by the previous student who was here). Into each titration flask, prepare 10 of acetate buffers according to the table: Solution of acetic acid (CH 3 COOH) and solution of sodium acetate (CH 3 COONa) are located in automatic burretes. How to use an automatic burret, it is desribed in section "a) Colorimetric determination of ph". Buffer number CH 3 COOH (100 mmol/l) CH 3 COONa (100 mmol/l) The total volume of the solution is the same in all the flasks, it is Also the total buffer concentration is exactly the same in all the flasks (c = 100 mmol/l). When ready, add 2-3 drops of indicator (phenolphtalein) into each titration flask with buffer solution you have prepared. The solution remains colourless. Fill the burette with standard solution of NaOH (c = mol/l) and titrate all 10 buffer samples until colour change of indicator. How to titrate? You should already know this procedure... If you do not remember: It is the best to hold titration flask in your right hand (and mix gently the content all the time). Use your left hand to control the stopcock of the burette. Add slowly (drop by drop) standard solution from the burette to the sample in titration flask. Watch the colour of the solution in titration flask all the time. Stop the titration (addition of the solution from the burette) in the "equivalence point" (sudden change in colour) the solution in titration flask turns pink colour in this case. Record the volumes of NaOH needed to reach the "endpoint" (table to put data into is enclosed). The column for data measured in the laboratory is marked in the table! The other columns of the table can be processed at home after the lab work is done. Calculate the amount of NaOH that caused the change in ph of the buffer solution (that caused the change in colour of an indicator in the titration). n(naoh) = c V(NaOH) Use Henderson-Hasselbalch equation to calculate ph of individual buffers. ph = pk a + log CH3COONa CH3COOH 5 pk a = 4.75

8 On a graph paper, plot the following values: axis x ph of individual buffer axis y amount of NaOH that caused the change in ph Mark into a graph all the ten points (by the crosses or the dots). Draw (with a colour pencil) the best fit and smooth curve between the points. Not all the points plotted in a graph must necessarily be on the curve! Make an approximation average by drawing your curve. How to draw a best fit smooth curve is demonstrated in the picture on hyperbolic curve. outlier point (probably erroneous measurement) No bent line, no waved line connecting the points! What type of curve is plotted in your graph? hint: it has a shape of certain "letter" For mathematical estimation of the maximum buffering ability, it is helpful to find "inflection point" of the curve. Inflection point is the point where the curve changes from being concave to being convex (from curvature upwards to curvature downward) or vice versa. To identify inflection point, we can use the knowledge that the first derivative has extreme value (local maximum or minimum) in inflection point. To plot the first differential function, calculate: (two columns on the right side of the table) ph 1 + ph 2 ph 2 + ph 3 ph n-1 + ph n,,..., (average ph of two neighboring samples) The differential (a change in n(naoh) with the respect to a change in ph): n NaOH / ph n(naoh) 1 - n(naoh) 2 n(naoh) 2 - n(naoh) 3 n(naoh) n-1 - n(naoh) n,,....., ph 2 - ph 1 ph 3 - ph 2 ph n - ph n-1 On a graph paper, plot the first differential function: axis x average ph of two neighboring samples axis y corresponding differential Draw (with a colour pencil) the best fit and smooth curve between the points. Find its maximum. Find the ph where the acetate buffer exhibits the best buffering properties: Make conclusions: 6

9 Table to put data into Buffer number CH 3 COOH (100 mmol/l) CH 3 COONa (100 mmol/l) ph V(NaOH) n(naoh) mmol ph n-1 + ph n 2 n(naoh) n-1 - n(naoh) n ph n - ph n Values measured in the laboratory 7

10 n NaOH (mmol) 1.20 The buffer effect of acetate buffer Graphical evaluation n NaOH / ph ph 8