ACIDIMETRY AND BUFFERS
Key words: Henderson - Hasselbalch equation, pH of buffer, function of buffer, concentration
of buffer, capacity of buffer, acetate buffer, phosphate buffer.
Reagents:
1. Acetic acid solution
1 mol/l (task 1.)
2. Hydrochloric acid solution
0.1 mol/l
3. Sodium hydroxide solution
1 mol/l
4. phenol red (0,1g dissolved in 60ml of ethanol and filled in 100ml with distilled water)
5. gastric juice samples
1. Quantitative titration analysis in medicine . Determination of gastric juice
acidity.
Determination of gastric juice acidity is still one of the basic investigating biochemical
reactions in gastroenterology. At present time, may be carried out in combination with
gastroscopy. Quantitative method for determining the acidity is titration of 0.1 mol / l NaOH,
the acidimetry.
Gastric juice is a clear colorless liquid, sometimes viscous from mucus. It has a characteristic
odor and strongly acid reaction, coming from the free hydrochloric acid (HCl). gastric juice
pH is in the range 0.8 - 1.5 , which corresponds HCl 140-150 mmol / l Swallowed food buffer
it to pH 1.8 - 4, which is optimal for the action of pepsin and gastric acid-resistant lipase .
Density of gastric juice varies between 1.001 to 1.010 g/ml. Regulation of gastric juice
secretion is neurohumoral. Production is in the range 1 000 - 3000 m1/24 hours
Gastric secretion is produced by three types of cells of gastric mucosa. Main cells form
pepsinogen, secondary, mucous cells produce mucus and protecting cells secrete H+, K+, Cl ..
The last cell type excludes HCl in concentration143 mmol / l. Hydrochloric acid is partially
neutralized after contact with the mucosal surface alkali mucin containing NaHCO3.
The acidity and amount of gastric juice are determined mainly by the number of cells that are
involved in the secretion.
An important factor is the level of the peptide hormone gastrin, produced by the cells of
gastric mucosa. Determining of hydrochloric acid output (i.e. the product of the substance
concentration of HC1 and the amount of gastric juice) measured at a certain time interval is
therefore important for the assessment of pathological changes in the gastric mucosa. This
indicator is measured both in resting basal conditions and after stimulation (provocation) of
gastric secretion to the maximum possible values. Histamine or synthetic hormone
pentagastrin are used tostimulation of gastric secretion.
Collection of gastric juice:
The patient comes to the examination in the morning after an overnight 12 hour fasting and
not smoking. In clinical practice, the gastric juice is gained by thin nasogastric tube, which is
inserted into the lowest part of the stomach cavity. The patient swallowing the probe can sit or
lie on his left side, the position of the probe is checked by sciascopy. Using 20ml syringe is
taken every content from stomach after starving, which is not evaluated, and then in 2-minute
intervals is collected gastric juice under light pressure.
During a two-hour sampling is under basal resting conditions, for a period of 60 minutes
obtained fractions of B. Then pentagastrin 6 mg / kg body weight) is subcutaneously injected
and for the next hour (l5min intervals) are obtained fraction S1 to S4.
Principle:
By volumetric analysis (titration with sodium hydroxide), acidimetry is determined the
concentration of HC1 and its output is evaluated.
· Working procedure:
- First you will notice the smell , color and consistency of gastric juice. Green or yellow color
shows admixture of bile, red or dark colour indicates blood admixture. Stomach contents with
bile admixture, in which pancreatic secretions are also presented, can not be quantitatively
evaluated by using HC1 (pancreatic juice contains hydrogen carbonate) .
- Measure the volume of fractions using the cylinder (100 ml)
- pipette accurately 5.0 ml of fraction to Erlenmeyer flasks (100 ml), add 3 drops of phenol
red and titrate with NaOH (concentration 0.1 mol / l; The factor is determined by
kaliumhydrogenftalátu 0.1 mol / l oxalic acid or 0.1 mol / L phenolphthalein). The
equivalence point is exhibited as a transition from yellow to red during titration of gastric
juice. If a red color appears immediately after addition of the indicator (before titration) , rate
NaOH consumption as zero .
Evaluation:
- Calculate HCl concentration in the fractions by the formula (B, S1- S4) :
mmol HCl in 1 liter of juice = a x 20
a = the consumption of 0.1 mol / l NaOH in ml (justify the calculation)
- Plot fraction volume and HCl concentration, coordinates are shown in Figure
- Calculate the basal secretion (output) HCl. Multiply the amount and HCl concentration of
fraction B. The product divided by 1000 is the basal HCl dispensing in mmol per 60 minutes
(normal value is 1.5 to 4 mmol/60 min).
- Multiply the volume and HCl concentration in the fractions 1-4 and divide the sum of the
products by 1000. This value represents the peak acid output (PAO).
- Calculate the maximum secretion of HCl (maximal acid out- put, MAO).
Fig. HCl titration in gastric juice - basal secretion and secretion following pentagastrin
stimulation
In clinical practice is as follows:
from fractions 1-4 are selected the two neighboring fractions, in which was achieved the
highest output (sum of the HCl concentration in mmol / l and the fraction volume in ml
divided by 1000). Both outputs are summed and multiplied by two (Justify the calculation).
Normal values of maximum and peak HCl secretion are between 10-23 mmol/60 min.
- Evaluate the real data about your patient.
The basal output greater than 15 mmol / h is diagnostically significant if suspected Zollinger Ellison syndrome (set of symptoms caused by a tumor or islet cell).
Patients with duodenal ulcer tend to have higher values of maximum secretion. In contrast
this diagnosis is unlikely, when basal achlorhydria (absence of hydrochloric acid in gastric
juice) and while secretion is stimulated less than 15 mmol / hr for men and 10 mmol / hr for
women. Achlorhydria during maximum stimulation test shows the disappearance of covering
cells, ie, diffuse damage of the stomach lining (atrophic gastritis). Detection of achlorhydria
with radiographically proven puncture of stomach lining lead to suspicion of cancer (gastric
cancer).
Normal levels of HCl output :
BAO ( Basal Acid Output) = basal supply of HCl on an empty stomach 1-5 mmol / hr
MAO (Maximal Acid Output) = total gastric secretion after stimulation during 60 min 13 to 25 mmol / h
PAO ( Peak Acid Output) = sum of two consecutive 15 minute fractions with the highest
secretion of HCl are multiplied by two (equivalent to 1 hour ) 0-34 mmol / hr.
Pathological values rating:
BAO : 1-5 mmol / h normal , gastric ulcer possible
5-15 mmol / h duodenal ulcer
> 20 mmol / h suspicion of gastrinoma ( Zollinger -Ellison syndrome ).
PAO : 0 mmol / h atrophy of the gastric mucosa (chronic atrophic gastritis , pernicious
anemia )
5-40 mmol / h normal , gastric ulcer possible
> 40 mmol / h suspicion of gastrinoma ( Zollinger -Ellison syndrome ).
BAO / PAO: < 0.20 mmol / h normal, gastric ulcer possible
> 0.60 mmol / h gastrinoma (Zollinger -Ellison syndrome).
2. Dependence of pH of the acetate buffer on the molar concentration ratio of
ethanoate (acetate) and ethanoic (acetic) acid.
Principle:
In the case of acetate buffer, the components are acetic acid (the weak acid, pKA = 4.75)
and its conjugate base, sodium acetate. According to Henderson - Hasselbalch equation, pH of
buffer depends upon the molar concentration ratio of both components. Make up buffer
solutions of given ratio [A-]/[HA] by adding different volumes of sodium hydroxide solution
to a given volume of acetic acid solution.
Procedure:
a. Pipette the solutions of 1 mol/l acetic acid, 1 mol/l sodium hydroxide and distilled water
into three beakers following table 1.
Table 1.
Beaker No
1
!2!
3
[A-]/[HA]
1:3
1:1
3:1
CH3COOH 1 mol/l (ml)
10
10
10
NaOH 1 mol/l (ml)
2.5
5
7.5
Distilled water (ml)
7.5
5
2.5
pH measured
pH calculated
[Buffer] (mol/l)
b.
c.
d.
e.
f.
Stir the solutions using glass rod and measure the pH values of prepared buffers using pH
- meter.
Calculate the pH values of prepared solutions.
Compare the measured pH values with calculated ones.
Calculate molar concentrations of prepared buffers.
Inscribe the results into table 1.
CARE! Buffer prepared in beaker ! 2 ! (ratio of components 1: 1) will you use at further tasks.
3.
The effect of adding acid to the acetate buffer.
Principle:
The added acid (hydroxonium ions) reacts with the acetate ions present in the buffer
solution forming undissociated acetic acid. The pH change is therefore again minimal.
Procedure:
a. Pipette the acetate buffer solution prepared in the task 1. (beaker !2!, ratio of components
1:1), distilled water and 0.1 mol/l hydrochloric acid solution (use burette) into two beakers
following table 3.
Table 3.
Beaker No
1
2
Acetate buffer (ml)
5
-
Distilled water (ml)
-
5
HCl 0.1 mol/l (ml)
5
5
pH measured
pH difference (measured)
pH calculated
pH difference (calculated)
b.
c.
d.
e.
f.
Stir the solutions using glass rod and measure the pH values of the prepared solutions.
Calculate the pH values of the solutions.
Compare the measured pH values with calculated ones.
Determine the pH change after addition of acid to the acetate buffer solution in contrast to
distilled water.
Inscribe the results into table 3.
Beaker No
1
2
Acetate buffer (ml)
5
2.5
Distilled water (ml)
-
2.5
[Buffer].(mol/l)
pH measured
pH calculated
!CARE ! Buffer prepared in the both beakers will you use at next task 5.