manual dri laboratory

D R A I N A G E R E S E A R C H I N S T I T U T E C D R H C A I R O E G Y R T

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DRAINAGE WATER

I N S T I T U T E F O R L A N D A N D W A T E R M A N A G E M E N T R E S E A R C H C I C W ) W A G E N I N G E N T H E N E T H E R L A N D S

MANUAL

DRI LABORATORY

M A N U A L DRI L A B O R A T O R Y

Manual Report

For

Chemical and Biological

Analysis

by

Drs.Joop Harmsen

Dr. Laila El Sissy

Dr. Garaal Abdel Nasser

Overview Reports Measurement Programme

MANUALS CALIBRATIONS (Measurements)

YEARBOOKS Chemical Data Hydrologicil Data

SPECIAL REPORTS

Manual Short Term Measurement Programme T

Accuracy Analysis Routine Measurement Programme 16

Manual Measurement Programme

17

Users Manual Da la Base System and Computer Programmes ^8

Manual DR I Laboratory

19

Description Measurement Network

20

Calibration Measurement Eastern Oetta 1981 - 1984

Calibration Measurement Western Delta 1981 - 1985

Calibration Measurement Middle Delta 1981 - 1985

Calibration Measurement Nile Delta Update 1986

Yearbook Eastern Delta 1980 - 1983

I Yearbook Western Delta 1980 - 1983

10

Yearbook Middle Delta 1980 - 1983

11

Yearbook Nile Delta 1984

13

Drainage Water Nile Delta 1 9 8 0 - 1 9 8 3 Discharges and Salinities ;

Estimation available Water El Salam Canal Project

Drainage Water Nile Delta 1984 Discharges and Salinities

12

Estimation available Drainage Water El Umum Reuse Project

X Nile Delta 1985 Discharges Salinities Chemical Data 14

21

Nile Delta 1986 Discharges Salinities Chemical Data 15

FINAL REPORT Measurement Programme J J

C O N T E N T S

1. Introduction. 4

2. Possibilities of the DRI Laboratory. 5 2.1. Chemical Analyses. 5 2.2. Biological Analyses. 6

3. Data management. 7 3.1. Description of the samples. 7 3.2. Analyses. 9

4. Theoretical background of some methods. 13 4.1. Conductivity. 13 4.2. Atomic Absorption. 14 4.3. Flame photometer. 25 4.4. Chloride Potentiometrie. 25 4.5. The determination of bicarbonate

and carbonate. 27 4.6. Oxygen demand. 29

4.6.1. Biological. 29 4.6.2. Chemical. 30

5. Working Procedures of Instruments on DRI. 31 5.1. Atomic absorption spectrophotometer. 31 5.2. Use of the diluter. 38 5.3. Spectrophotometer Spectronic 21. 39 5.4. Oxygen - electrode. 39 5.5. Calibration of Sartorius 2002 MP 1. 40

6. Chemicals and solutions. 41 6.1. Preparation of titration solutions. 41 6.2. Standard solutions. 41 6.3. Adjusting normalities. 42 6.4. pH. 44 6.5. Conductivity. 44 6.6. Chloride. 44 6.7. Bicarbonate. 44 6.8. Calcium and Magnesium. 44 6.9. Calcium. 45 6.10 Sulphate. , 45 6.11 Sodium and potassium. 45 6.12 Nitrate. 45 6.13 Nitrite. 46 6.14 Biological oxygen-demand. 46 6.15 Chemical oxygen-demand. 47

7. Description of methods used on DRI. 48 7.1. pH. 48 7.2. Conductivity. 51 7.3. Titrations. 54 7.4. Chloride. 55 7.5. Bicarbonate and carbonate 58 7.6. Calcium and Magnesium 60 7.7. Calcium 61 7.8. Sulphate 62 7.9. Sodium and Potassium 65 7.10 Photometric determination of nitrate 66 7.11 Photometric determination of nitrate 68 7.12 Bacteriological analysis 69 7.13 Biological oxygen - demand. 75 7.14 Chemical oxygen - demand. 77

8. Maintenance of instrumentation. 78 8.1. General. 78 8.2. Maintenance of spectronic 21. 79 8.3. Maintenance of electrodes. 80 8.4. Atomic absorption spectrophotometer. 80 8.5. Maintenance laminair flow bench. 81

9. References. 82

Annex. Description of methods in the Arabic language.

1.1 N T R O D U C T I O N

In the Reuse of Drainage Water Project the DRI laboratory has an important task. In the measuring program about 250 samples are taken to be analysed. Results of the analyses are used for further calculations. To assure the quality of the results the people working on laboratory have to be well trained. They have to use well documented methods and well functioning equipments. Methods used on the laboratory are well known and mostly described in the standard methods. In this manual these methods are presented on such a way, that they can be used with the specific instrumentation available on the DRI-laboratory. For some methods theoretical backgrounds are presented. Methods used have to be calibrated on a proper way and it must be possible to check the calibration. Therefore the laboratory uses reference samples, check on calculations etc. which are described in this manual. Every instrument will fail when it is not kept under maintenance. This can be done by an service organization, but such an organization is not always present and mostly very expensive.However most of the maintenance can be done on the laboratory and the way to do it is described in the manual.

At the moment techniques are changing very quickly and computers are used more and more. Therefore the effective lifetime of the this manual will be short because alaboratory has to work with a manual which is up to date. The complete manual is therefore also present on floppy disk with the head of the laboratory,she has the authority to make changes in the manual. The changes have to be made in all the distributed manuals. At least every two years a new version of the manual has to be distributed. This version of the manual is valid till January 1991.

2 .P O S S I B I L I T I E S O F T H E D. R. I .

L A B O R A T O R Y .

On the DRI l abo ra to ry , i t i s p o s s i b l e t o conduct chemical , b i o l o g i c a l and p h y s i c a l r e s e a r c h . This manual d e a l s w i th t h e chemica l and b i o l o g i c a l d e t e r m i n a t i o n s . In o r de r t o do t h e work , l abo ra to r i e s and equipments a re a v a i l a b l e as descr ibed h e r e .

2 . 1 CHEMICAL ANALYSIS.

In the chemical p a r t of the l abora tory , samples are analysed for s e v e r a l parameters as p resented in t a b l e 1. Use i s made ofmodern equipments l i k e pH/mv-meters , c o n d u c t i v i t y m e t e r s , m o t o r b u r e t s , s p e c t r o p h o t o m e t e r s a f l amepho tome te r , K j e l d ah l equipment and an a tomic a b s o r p t i o n s p e c t r opho t ome t e r . A l so e q u i p m e n t s l i k e a n a l y t i c a l b a l a n c e s , s h a k i n g m a c h i n e s , c e n t r i f u g e s and t he n e c e s s a r y g l a s s w a r e and c h em i ca l s a r e a v a i l a b l e . The d i s t i l l e d water i s suppl ied with a d i s t i l l e d w a t e r a p p a r a t u s . The i n s t r u m e n t s u s ed f o r a c e r t a i n de terminat ion are a l s o given in t a b l e 1. The l a s t column in the t a b l e g ives the p r i n c i p l e of the method used. Some parameters can b e a n a l y s e d w i th d i f f e r e n t methods , which i s impo r t an t when one f a i l s .

Table 1. Methods of t he chemical l a bo r a t o ry .

Parameter

pH Conductivity Chloride Chloride Bicarbonate Bicarbonate Sulphate Sodium Sodium Potassium Potassium Magnesium Magnesium Calcium Calcium Ammonium Nitrate Nitrite

Instrument

pH-meter conductivity-meter motorburet idem + mV-meter motorburet idem + pH-meter Spectrophotometer Flamephotometer AAS Flamephotometer AAS motorburet AAS motorburet AAS Kjeldahl dest. Spectrophotometer Spectrophotometer

Principle

Electrode Electrode Titration Titration/electrode Titration Titration Turbidity Atomic emission Atomic absorption Atomic emission Atomic absorption Titration Atomic absorption Titration Atomic absorption Titration Color development Color development

2.2 BIOLOGICAL ANALYSIS.

On the biological laboratory analysis are carried out in which micro-organism are involved. These are; BOD (Biological Oxygen Demand), MPN (Most Probable Number). Also the COD (Chemical Oxygen Demand) is determined in this section. All the equipment necessary to do these analysis is available on the laboratory as listed in table 2.

Table 2. Equipments on the biological laboratory.

Parameter Equipment

COD COD-equipment,Digital buret BOD Oxygen electrode Test on micro-organism Steriliser,pump for media,plate

counter,sterile filtration units, laminair flow bench, ice box.

For microbiological work it is very important to handle samples in a sterile environment. Otherwise not the microorganism in the samples are determined, but those in the environment. During preparation of the media, micro-organism may enter the solutions. This is no problem, because all plates and tubes are sterilized before use. When the samples are inoculated in the media it is important to work sterile. With this work a laminair crossflow bench is used. In this bench air is filtrated over a very fine filter (HPA filter system). Micro-organisms and spores cannot pass through this filter. The air is blown over the working area with such a velocity that dust and micro-organisms cannot enter the bench. If the bench is cleaned with alcohol before use a sterile environment is obtained.

D A T A M A N A G E M E N T .

The computer is used more and more for management of data. Also on the DRI laboratory the computer will have an important place. Use of it on the laboratory is however just started and therefore a complete manual for data management cannot be given now. Introducing of a computer takes time and cannot be forced. That is why only a description is given how things have to be arranged. Partof it is done already by computer andthe rest of it by hand.

Taking in mind the experience of other laboratories it is important to use standard software like D base and Lotus. Development of own software must be restricted as much as possible. For a good data management everything between ordering of sampling and the final data have to be described. A good cooperation between the laboratory and the project team is necessary. The whole system is primary made to prevent mistakes. Secondary it gives possibilities to check all steps, if there is doubt about the results. How things are organized as presented in fig 1.

3.1 DESCRIPTION OF SAMPLES.

The samples are received on the laboratory by the responsable engineer. There must be a form on which all the relevant information of the samples is written as, parameters to be analysed, sample codes, EC-field and special remarks necessary to improve the results. Samples must have a code and a date,as for instance MGl 03/06/87. The code may have 5 characters. These codes are entered into the computer together with the EC measured in the field. Also a laboratory series and analyse number is entered and written on the bottle as RA3 (Re-use series A sample 1). To the computer a list is given with the parameters to be analysed. From the computer a datasheet is obtained to use in the laboratory. On this sheet check samples and questions about calibrations of the instruments are included. The samples and datasheets go to the engineers responsible for a certain project.

or eng. or eng. or eng. or eng. or eng.

Re-use Pilot areas Payum EPAD Others

eng eng eng eng eng

Information, data Samples Check

Project teams

c/rc^st3n- —

Sampling what and where

Sampling, EC measurement, codes, specific circumstances

/

CO' tP

SP' ^ ^

.«*

Laboratory

V; ';\

\ \

Entering of samples in laboratory data system

5

Measurements

_L

1 r Original data

1 L Check storage of data — check methods - f i e l d EC — L a b EC — Calc. EC ~ ~ Lab EC

— sum anions —»sum cations

• are special circumstances responsible for " strange results"

I Data + comment

Codes, EC

Previous data

Check data by project team and laboratory — check codes — are their sudden changes in result, for instance

in TDS/EC

1 Final data

Data bases — laboratory - project teams

Fig 1. Management of samples and data.

3.2. ANALYSIS.

On the datasheet all original results as titration volumes, normalities and sample volumes are written. These values are entered into the computer together with answers on the questions. The program is developed by Dr. Abdelklalik to calculate the chemical analyses. From the original results it gives the cation and anion concentration in meq/1. At the moment it calculates the sulphate concentration by difference and it calculates water quality parameters as SAR, adj-SAR, RSC and TDS. The EC is also calculated and compared with the measured EC. If the results are accepted, the program automatic transfers the data to D-base. The most easy way is to enter the original results as sample volumes, titration volumes and readings from instrument. The program has an option to enter calculated results. Only the head of the laboratory is allowed to make changes in the program in close cooperation with Dr. Abdelkhalik.

In the case that a method is well calibrated it should give the same results analysing one sample several times. The results of such a sample, a reference sample, can be used to assure a constant quality of the laboratory. On the DRI laboratory 2 reference samples are present, one is representative for water samples, the other for soil paste samples. The results are registered on a control chart as shown in figure 2.

I 0 r

2 u

• 17 8

•_5% deviation

• 3V« deviation

•' A %• . - . _ ! ^ • H\ \ *-3% ist

deviation

5% deviation

' i i i i _ _i i 1 J F M A M j J A S O N D J F

fig 2. Control chart used for chlorid determination in water

With t h i s cha r t i t i s p o s s i b l e t o p revent big mistakes. If e v e r y t h i n g i s c o r r e c t , a c o n s t a n t v a l u e between t he two 3 % l i n e s i s o b t a i n e d . 3 % i s t h e d e v i a t i o n , t h a t w i l l be a c c ep t e d . R e s u l t s between t h e 3 % and 5 % l i n e s a r e a l s o accepted, but the method must be screened. Devia t ions of more t h en 5 % a r e not a c c ep t ed . A r ea son must be found, b e fo re t h e method i s used for a no the r s e r i e s of s amp le s . Samples a n a l y s e d in t h e same s e r i e s h ave t o be r e a n a l y s e d o r r e c a l c u l a t e d . The l a t t e r i s on ly a l l owed i f t h e m i s t ake was caused by a m i s t ake in c a l c u l a t i o n or fo r i n s t a n c e a wrong normal i ty . In the f igure few examples are g iven:

A The r e s u l t s are normal d i s t r i b u t e d , a l l r e s u l t s are accepted. B A d r i f t in the r e s u l t s i s v i s i b l e . When the d ev i a t i on

was in t h e r eg ion between 3 and 5 % i t was found t h a t t h e n o r m a l i t y of t h e AgN03 was changed. C o r r e c t i o n gave t he r i g h t v a l u e aga in and t h e same c o r r e c t i o n was done for t h e samples.

C A wrong sample volume was used. Reca l cu l a t i on gave the r i g h t v a lue .

D In t h i s c a se no s p e c i f i c r e a son was found. A l l s o l u t i o n s were renewed and t h e r e s u l t s were good a g a i n . Because no c l e a r r e a son was found, a l l s amples of t h e same s e r i e s were r eana lysed .

Carbonate and b i ca rbonate

Sometimes, e s p e c i a l l y when wastewater i s flowing i n t o the d ra in , i t may occur t h a t o ther bas ic components are p r e sen t . In t h a t case normal c a l c u l a t i o n s w i l l g ive wrong r e s u l t s , because t h e b i c a r b o n a t e c o n t e n t s i s much t o h i gh . I t i s however p o s s i b l e t o r e c o g n i s e t h e wrong r e s u l t s . From chemica l e q u i l i b r i a the fo l lowing r e l a t i o n i s obta ined.

pH = 10.38 + log[C03(2-)] / [HC03(-) ] [ ] = concen t ra t ion in meq/1

Th is r e l a t i o n i s a l s o p r e s e n t e d in f i g u r e 3. I f a m i s t ake in t h e pH of 0.1 u n i t or a m i s t ake in [C03 (2 - ) ] / [HC03(-)] of about 20 % i s a c c e p t a b l e , a r e g i on in f i g u r e 3 i s obta ined in which good r e s u l t s have to f i t . If the r e s u l t s of t h e a n a l y s i s g i v e a p o i n t below t h e r e g i on o t h e r b a s e s can be p r e sen t . F i r s t the a n a l y s i s must be checked and i f the r e s u l t s a r e t h e same t he f o l l o w i n g c a l c u l a t i o n s must be u sed . In t h i s c a l c u l a t i o n i t i s assumed t h a t t h e amount t i t r a t e d between pH 8.3 and 4.1 i s a measure for the inorganic carbon content and t h a t a l l the o ther bases are t i t r a t e d above pH 8 . 3 .

10

[C03] = 20 x n x (b-a) x f(C03) meq/1

[HC03]= 20 x n x (b-a) x f(HC03) meq/1

amount of other bases = 20 * n * a - [C03]/2 meq/1

n = normality of HCl

a = amount of HCl used to reach pH 8.3

b = amount of HCl used to reach pH 4.1

f(C03)= fraction of C03 present at the pH of the sample.(see table 3)

f(HC03)= fraction of HC03 present at the pH of the sample.(see table 3)

If more or less than a sample volume of 50 ml is used, multiply with; 50/volume of sample (ml)

Table 3.Fractions of C03 and HC03 as a function of pH. The concentrations are in meq/1. This table can be used if other bases are present in the sample.

pH f ( C 0 3 ) f (HC03)

8 . 3 0 . 000 1 .000 8 .4 0 . 0 2 1 0 . 9 79 8 . 5 0 . 0 26 0 . 974 8 . 6 0 . 0 32 0 . 9 6 8 8 . 7 0 . 0 40 0 . 9 60 8 . 8 0 . 050 0 . 9 50 8 . 9 0 . 0 62 0 . 9 3 8 9 . 0 0 . 0 77 0 . 9 2 3 9 . 1 0 . 0 9 5 0 . 9 0 5 9 . 2 0 . 117 0 . 8 8 3 9 . 3 0 . 1 4 3 0 . 857 9 . 4 0 . 1 7 3 0 . 826 9 . 5 0 . 2 09 0 . 7 9 1 9 . 6 0 . 2 4 9 0 . 7 5 1 9 . 7 0 . 2 9 5 0 . 7 0 5 9 . 8 0 . 3 4 5 0 . 6 5 5 9 . 9 0 . 3 98 0 . 6 0 2 1 0 . 0 0 . 4 5 5 0 . 5 4 5

11

10.0

figure 3. Relation between log [C03(2-)]/[HC03(-)] and the pH. Points between the dotted lines are accepted.

12

4 . T H E O R E T I C A L B A C K G R O U N D O F

S O M E M E T H O D S .

4 . 1 CONDUCTIVITY.

Conduct iv i ty i s the numerical express ion of the a b i l i t y of an aqueos s o l u t i o n t o c a r r y an e l e c t r i c c u r r e n t . This a b i l i t y depends on t h e p r e s en c e of i o n s , t h e i r t o t a l c o n c e n t r a t i o n s , m o b i l i t y , v a l e n c y and r e l a t i v e c o n c e n t r a t i o n s , and on t h e t e m p e r a t u r e of t h e s o l u t i o n s . The c o n d u c t i v i t y can be expressed with the equat ion;

K(t)= c ( i ) . a ( i )

K(t) = conductivity.

c(i)= specific conductivity of ion (see table 4.)

a(i)= activity of ion 'c'.

Table 4.Specific conductivity of ions commonly found in water. Conductivity expressed in uS/cm = umhos/cm (Standard Methods 1980).

ION SPECIFIC CONDUCTIVITY (per meq/L)

48.9 72.0 52.0 46.6 75.9 73.9 43.6 84.6

Na K Ca Mg Cl S04

( + ) ( + ) (2+) (2+) ( - ) ( 2 - )

HC03(- ) C03 ( 2 - )

13

4.2. ATOMIC ABSORPTION.

4.2.1. T h e o r y

Atomic absorption is an analytical technique in which compounds in a sample are dissociated into atoms in gaseous form. The atoms are in the ground state which means that they are able to go to an excitated state by absorption of light.

M + hv MH

M=metal in ground state

hv=light with energy hv

M*=metal in excitated state

If light with an energy of hv is passing the gaseous atoms, there will be absorbtion of light. The fraction of light absorbed is a measure for the concentration of the metal in the sample.

atoms in excitated state

hv

•> atoms in ground •> state

light with energy hv Intensity (Io)

•> Transmitted •> radiation

Intensity (It)

TheTransmittance It / Io can be represented by Beer's Law.

It - a b c = e

Io

a= absorption coefficient (different for every b= length of absorption path c= concentration of absorbing atoms.

element)

14

The absorbtion A is defined as: - log(It/Io)

So A = 1/2.3*a b c

With a c o n s t a n t p a t h l e n g t h and f u r t h e r a l l i n s t r u m e n t condi t ions cons tan t .

A = cons tant * c

4 . 2 . 2 . T h e i n s t r u m e n t .

The b a s i c components of an a tomic a b s o r p t i o n s p e c t r o photometer are given in f ig 4.

| source of| | a tomis ing | | monochro-| | de tec | | r e g i - | j r ad ia t ion j> j system j>| mator |>j t o r j > j s t r a - j I I I I I I I I I t i o n | I I I I I I I I I I

f i g . 4 Diagram of an atomic absorpt ion spectrophotometer .

SOURCE OF RADIATION:

E v e r y e l e m e n t h a s a s p e c i f i c w a v e l e n g t h a n d s p e c i f i c h o l l o w c a t h o d e lamp . The l amp i s made of t h e m a t e r i a l , we want t o m e a s u r e . I n t h e l amp t h e f o l l o w i n g r e a c t i o n o c c u r s :

M* = M + hv

D i f f e r e n t h o l l o w d i f f e r e n t e l e m e n t s .

c a t h o d e l a m p s h a v e t o b e u s e d f o r

ATOMISING SYSTEM.

With flame atomic absorp t ion , the sample i s a s p i r a t ed in a f lame. By the high temperature the water evapora tes and the molecules are d i s soc i a t ed i n t o atoms, which a re ab le t o absorb l i g h t . The sample i s a s p i r a t ed i n t o the flame with the a id of a n ebu l i s e r . In t h i s p a r t the sample i s sucked and mixed with the flame gases . After t h a t i t l e aves the burner head through a l ong (10 cm) narrow s l i t , and a l l t h e s a l t s a r e a tomised by t h e h igh t empe r a t u r e in t h e f l ame . The t empe r a t u r e in t h e a c e t y l e n e / a i r flame i s between 2350 and 2450 'C.

15

For a stable reading and a good sensitivity the following flame factors are important.

) or - Composition of the flame, oxidizing (excess of air reducing (excess of acetylene).

- The density of atoms is not constant in the flame. So optimum height has to be searched. This height is also influenced by the composition .

- If high saline water is aspirated the burner head can be clocked with salts, thereby reducing the sensitivity or enhancing the noise.

Sometimes other flame types are used, for instance acetylene-nitrous oxide. This flame has a higher temperature, thereby reducing chemical interferences. Most compounds however can be measured with an acetylene-air flame. If a higher sensitivity is needed a graphite furnace can be used as an atomising source. This furnace is often used for measurements of low concentrations of heavy metals.

MONOCHROMATOR.

The monochromator is used for selecting the line to be measured with acid of one or more gratings. This has to be done very accurate. After a first course adjustment the optimum has to be searched. This is the point where maximum light reached the detector (i.e. maximum trans-mittance or minimum absorbance). On the monochromator also the slit has to be selected. For every element and wavelength the optimum is recommended by the manufacturer of the instrument. The value depends on how many lines a lamp gives. For instance calcium gives a line at 422.67 nm (fig.5) in this neighbourhood no other lines exist, and a high slit width can be used. Iron however has in the neighbourhood of the line at 248.33 nm also other lines (fig.5). By selecting a small slit these other lines cannot reach the detector and better results are obtained. Therefore the recommended slit values achive improve the results.

16

® Slit 07 nm I I 422.67

424 421 250 X I nm I

fig.5 A emission spectrum great slit width possible

B small slit width to exclude the non absorbing line.

DETECTOR.

The d e t e c t o r i s a p ho t ome t r i c t u b e . R ad i a t i o n from t h e monoch roma to r w i l l f r e e e l e c t r o n s on t h e p h o t o c a t h o d e . By c o m b i n a t i o n of s e v e r a l e l e c t r o d e s t h e amount of e l e c t r o n s i s m u l t i p l i e d w i t h f a c t o r of 10*6 . The r e s u l t i n g e l e c t r i c a l c u r r e n t i s p r o p o r t i o n a l t o t h e amount of r a d i a t i o n . The l i n e a r r a n g e of t h e d e t e c t o r i s v e r y h i g h and c an be r e g u l a t e d by v a r i a t i o n o f t h e t o t a l p o t e n t i a l d i f f e r e n c e b e t w e e n t h e pho toca thode and t h e l a s t e l e c t r o d e (anode) .

REGISTRATION.

The s i g n a l from t h e d e t e c t o r i s t r a n s l a t e d i n t o t r a n s m i s s i on , absorbance or c o n c e n t r a t i o n u n i t s . I t i s p o s s i b l e t o c a l i b r a t e t h e i n s t r u m e n t w i t h s t a n d a r d s o l u t i o n s , b y which r e s u l t s d i r e c t l y i n c o n c e n t r a t i o n u n i t s a r e o b t a i n e d .

17

4.2.3. Description of the atomic absorption spectrophotometer

The atomic absorption spectrophotometer (A.A.S.) of the D.R.I, is a double beam type. This means that the light is splitted into two beams. One is passing the flame and the other one is used as a reference beam (see fig.6). With the chopper the light is modulated with a certain frequency. When the sample beam is interrupted, the reference beam reaches the detector and opposite. The detector is able to distinguish both beams and they can be brought together again after the flame. In the monochromator the right wavelength is selected with aid of a grating. After that the light is detected on the photomultiplicator.

Fig.6 Optics of AAS

THE LAMP

The lamp has to be placed in the lamp holder. The black knobs have to be between the mica spacers of the lamp. Alignment of the lamp can be done with the two black knobs. With [lamp] control on the front of the instrument, the lamp J-can be adjusted. With the [SIGNAL] switch on [LAMP] can be read on the meter. The current has to be current printed on the lamp.

current the current

the continuous

18

THE GAS SUPPLY.

The gases are supplied with the interlocked gas control system. This system has to give constant gas flow and constant pressure. Good measurements can be done however when the pressures of the gases are constant before the system. For acetylene this is right, because this is delivered in a gas bottle. When the pressure is corrected after ignition, it will be constant. The air is delivered by a compressor. The pressure of the system varies between 4 and 6 atm. (60 and 80 psi). To have a constant pressure a low pressure reducer is installed between the compressor and the A.A.S. The reducer is set on 3 atm. To prevent dust entry the A.A.S. a dust filter is used. In fig. 7 it is shown how the dust filter must be used.

Out

Cotton for filtering of dust

fig 7. Gasflow in the dust filter.

The cotton has to be periodicaly replaced.

THE NEBULIZER SYSTEM.

The nebulizer has to make a spray of the sample and mix it thoroughly with the gases. For a good and reproducable sensitivity the nebulizer has to be adjusted very well. This can be done by aspirating a copper solution of 10 ppm after preparing the instrument for measurement of copper.Then aspirate the solutions, loosen the locking ring and slowly turn the knurled nebulizer knob counterclockwise (fig.8). Stopturning when the nebulizer air blows back into the solution (small bubbles will appear at the end of the capillary tubing). Keep the end of the capillary in the standard and slowly turn the knurled knob clockwise. Watch the display while turning the knob ('SIGNAL'control on cont.). The reading will increase as aspiration begins. Adjust at maximum reading. At the end turn the locking ring against the knob to lock the setting.

19

© © ©

Fig.8 The nebulizer , exploded view.

THE FLAME.

The flame has to be exactly in the light beam. For this reason the horizontal adjustment (left knob) has to be on 8. The vertical adjustment must be on 7 or less. Higher settings of the vertical adjustment will cause a blockade of the light beam by the burner head. With a small piece of paper it can be controlled if the light is passing the slit in the burner head. A good flame is obtained with the air flow on 55 and the fuel on 32. For getting a higher sensitivity small adjustments may be necessary.

MONOCHROMATOR.

The wavelength can be adjusted with the [COARSE] adjust and the [FINE] adjust. With the [FINE] adjust the maximum on the [lamp/energy] meter has to be searched. i.e. Maximum light has to reach the detector. With the slit width the spectral bandpath can be set. With a wavelength of 400 nm, a slit width of 2 nm means that light from 398 to 402 reaches the detector. A slit width of 0.2 nm means that light from 399.8 to 400.2 nm reaches the detector. The slit width is important when the lamp also gives lines with light that cannot be absorbed.

DETECTOR.

The detector is a photomultiplier. The signal from the photomultiplier can be changed by the potential. If [SIGNAL] is on [SET UP] the potential can be changed with [GAIN] to have a reading on the [lamp /energy] meter. When the [SIGNAL] control is on the [ABS] or [CONC] position, this manual control is overriden by the automatic gain control.

20

REGISTRATION.

The i n s t r u m e n t i s c a p a b l e t o g i v e t h e r e a d i n g i n a b s o r b anc e - , c o n c e n t r a t i o n - and emiss ion u n i t s . I t can do t h e r e ad i ng on d i f f e r e n t ways (mode).

CONT.

The r e a d i n g i s i m m e d i a t l y c h a n g i n g when t h e f l a m e p r o p e r t i e s changes . I f t h e f lame i s c o n s t a n t t h e l a s t d i g i t i s changing because of t h e n o i s e of t h e i n s t r umen t .

HOLD.

If the [READ] knob is pressed, the instrument is taken the average value during 0.5 sec. This value can be set on values from 0.5 to 20 sec. with the numerical keyboard. The average value is more accurate than the continuous value because it eliminates a great part of the noise.

PEAK HEIGHT(HT) AND PEAK AREA.

The» pnak height and nron have to be used with a graphite furnace. Because this is not on the instrument, it is not to be used. With aid of the keyboard, orders can be given to the instrument.

AZ.

I s t h e a u t o z e r o . When a s p i r a t i n g a b l a n k . The i n s t r umen t w i l l g i v e a z e r o v a l u e a s soon a s t h e [AZ] i s p r e s s e d . The i n s t r u m e n t d o e s i t by c h a n g i n g t h e p o t e n t i a l o v e r t h e p h o t o m u l t i p l i e r . The [AZ] w i l l no t f u n c t i o n .

(1) if the [SIGNAL] switch is on [SET UP] or [LAMP].

(2) if a keyEntry is pending (the minus [-] will be flashing on the display)

21

[ T ]

I n t e g r a t i o n t ime key. Un less i n s t r u c t e d o t h e rw i s e t he i n s t r umen t u ses a 0.5 s e c . i n t e g r a t i o n t ime . To change the i n t e g r a t i o n t ime , e n t e r t h e t ime i n t e r v a l d e s i r e d , from 0.5 t o 20 sec. using the numerical keyboard. Then depress the [T] key. The i n t e r v a l s e l e c t ed w i l l be s to red in the instrument and used fo r each measurement u n t i l a d i f f e r e n t i n t e r v a l i s e n t e r e d or the instrument i s shut off. For normal a n a l y s i s an i n t eg r a t i on t ime of 3 s e c w i l l g i v e a c c u r a t e r e s u l t s . When low c o n c e n t r a t i o n s have t o be measured and t he absorbance i s near zero an i n t eg r a t i on time of 10 or 20 sec. can be n e ce s s a r i l y .

SI AND S2.

(Standard no 1 and standard no 2) keys. The keys are used to calibrate the instrument to display measurements results directly in terms of concentration. With the mode switch on [HOLD], the first standard is measured. After the measurement is complete the concentration value is entered and [SI] is pressed to set the measurement results equal to the desired concentration. [S2] is established on the same manner. The value for S2 can be changed without disturbing the value set for SI, but if Si is recalibrated, S2 must be recalibrated also.

The following conditions will prevent proper functioning of the [SI] and [S2] keys.

- If the [SIGNAL] switch is set on [ABS]. - The error character [E] will appear and the [-] will continue

to flash at the end of the measurement because;

a) The result is less than the zero set with the blank.

b) The expansion factor required to calibrate is greater than 50 x.

For instance with ignoring the decimal point, an absorbance of 0.010 cannot be displayed as 600 but has to be 60. The instrument automaticly drops as many digits to the right of the decimal point are necessary to bring the expansion within limits (A value of 10.00 will become 10.0 )

c) If the value entered for S2 is bigger than possible on the display > 9999.

d) If the value of S2 is lower than the value of Si.

The instrument also has a recorder output. This output signal is controlled by the [RECORDER] control switch.

22

[ABS] The signal send to the recorder is in absorbance unit.

[DUPL] Now the output is controlled by the [MODE] control. When scale expansion is used, it is also on the recorder signal. When the [MODE CONTROL] is on, hold the recorder pen will remain at rest until the completion of a read cycle. With [MODE] on hold a continuous signal is recorded.

[TCI], [TC2] and [TC3] Time constant setting, in these settings a continuous signal is send to the recorder with different damping values. [TCI] is a low damping (time constant 0.4 sec.) [TC2] and [TC3] gives more damping of the signal with time constants of respectively 1 and 3 sec.

4.2.4. M e a s u r e m e n t s ,

CALCIUM.

With c a l c i um some chemica l i n t e r f e r e n c e may o ccu r , Calcium r e a c t s w i t h p h o s p h a t e and g i v e s Ca3 (P04 )2 , wh ich compound i s o n l y p a r t l y d i s s o c i a t e d in t h e f l ame . So i f t h e r e i s phospha te i n t h e s a m p l e , t h e r e s u l t s w i l l be l o w e r . T h i s c a n be c o r r e c t e d , by a d d i n g an e x c e s s of 1 a n t h a n i u m c h l o r i d e t o t h e s a m p l e . L an t h an i um r e a c t s w i t h t h e p h o s p h a t e i n s t e a d of t h e c a l c i u m , by which a l l t h e c a l c i u m i s a b l e t o a t o m i s e i n t h e f l ame . L an than iumch lo r i de s o l u t i o n : d i s s o l v e 115 g LaC13 in 1 l i t e r 1 m o l / 1 HCl. SrC12 can a l s o be u s e d (180 g i n s t e a d of 115 g l a n t h a n i u m c h l o r i d e ) . The m e a s u r i n g s o l u t i o n s h a v e t o c o n t a i n 5 ml L aC13 - so l u t i on in 100 ml .

Magnesium

A l s o f o r magnesium LaC13 ha s t o be add t o t h e s o l u t i o n . The wave l eng t h 285.2 nm off magnesium i s v e ry s e n s i t i v e . The s e n s i t i v i t y can be r e d u c e d a l i t t l e by t u r n i n g t h e b u r n e r . I t i s a l s o p o s s i b l e t o t a ke a l e s s s e n s i t i v e l i n e , for i n s t a n c e 202.5 nm.

Sodium and P o t a s s ium

The f l a m e p h o t o m e t e r g i v e s good r e s u l t s , s o a t o m i c a b s o r b t i o n i s not n e s e s s a r y fo r sodium and po t a s s i um. However i t i s good t o know t h e a t o m i c a b s o r p t i o n me thod , f o r c h e c k i n g t h e f l a m e p h o t o m e t e r o r i n c a s e of f a i l u r e o f t h e f l a m e pho tome t e r .

23

With atomic absorption the temperature of the flame is very high. This high temperature causes ionisation because of the low ionisation energy of sodium and potassium.

Na = Na+ + e K = K+ + e.

The ions cannot absorb light. The two equilibria also influence each other. A high amount of sodium will drive the equilibrium of potassium to left, because of the high amounts of electrons from sodium. This change of equilibrium will give a higher absorption of light. It is possible to drive both equilibria to the left. Then all sodium and potassium atoms can absorb light. This can be done by adding an excess of cesium. Cesium ionise very easy and gives a high amount of electrons in the flame.

Cs = Cs+ + e.

With this high amount of electrons sodium and potassium are present as atoms.

HEAVY METALS.

For the heavy metals the samples and standards have to be acidefied. Sometimes back ground correction is necessary. Back ground absorption occurs with very saline or dirty water. The high amount of molecules will not dissociate completely in the flame. The molecules also absorb light which is called non atomic absorption. It is possible to correct for the non atomic absorption with aid of a hydrogen lamp. The hydrogen lamp gives a continuous spectrum instead of the line spectrum of a hollow cathode lamp. The principle of correction is given in fig 9.

100 % Hollow cathode lamp Hydrogen lamp

Light emitted

100% non atomic lOVc

atomic 20%

Silt width Silt width

Light absorbed

Fig 9. Signals measured with hollow cathode lamp and hydrogen lamp.

24

With the hollow cathode lamp 30 % of the light is absorbed. With hydrogen lamp slightly more than 10 % is absorbed. The difference of the two lamps gives the right value of 20 % absorption.

4.3 FLAME PHOTOMETER.

In a flame photometer samples are also aspirated into a flame. In the meter a propane/air flame is used, which has a lower temperature compared to the acetylene / air flame. Most elements are not completely atomised in the propane / air flame. This is not true for the alkalimetals. Salts of alkali metals are completely dissociated and the alkali metals are completely atomised. Due to the "low" temperature ionisation is not an important factor and addition of cesium is not necessary (compare 4.2.4). The outer electrons of alkalimetals are easely ecitated by the temperature of the flame. By returning to the ground state they emit light. This light can be measured. For sodium the yellow light of 589 nm is measured and for potassium the red light of 770 nm is measured. The correct wavelength is selected with aid of a filter.

4.4 CHLORIDE POTENTIOMETRIC.

Chloride is determined by titration with silvernitrate

Cl(-)+ Ag(+) > AgCl

The endpoint can be potentiometrically determined with the acid of a silverelectrode or with an indicator. The potential of the silverelectrode depends on the silverconcentration in the solution(Nernst equation).

E = Eo + 0.059 log[Ag(+)]

E=potential silverelectrode (V).

Eo=standard potential (at[Ag(+)]=1)

t]=concentration (mol/1)

In the beginning of the titration the Ag(+) concentration will remain low, because almost all of Ag(+) will precipitate as AgCl. The potential of the silverelectrode will only change slightly. Upon reaching the end point, the Ag(+) concentration w i l l increase sharply resulting in a big change of the potentiaKfig.10). The endpoint of the titration is that point where the change in potential by adding AgN03 is maximum. With the titration of Cl(-) the endpoint is always at the same potential, so the titration can be stopped when that potential is reached. For a saturated K2S04 reference electrode this endpoint of the titration is at -120 mV.

25

-300

-200

i

-100

120mV V - — E n d point

2 3 Volumelml AgN0 3 )

fig.10 Potential of the si 1 verelectrode as a function of added AgN03(reference saturated K2S04 electrode).

O

ri IU

UMI

c 3 0

o o o HIJ

o

O

O

O

fig.11 Experimental set-up.

(1) pH/mV m e t e r . (2) s i l v e r e l e c t r od e (a s i l v e r wire 2 mm). (3) S a t u r a t e d K2S04 r e f e r e n c e e l e c t r o d e . I t i s not a l l owed t o

use a calomel e l e c t r ode . This e l e c t r ode con ta ins Cl(-)which can en te r the s o l u t i on .

(4) Magnetic stirrer. (5) Metrohm buret.

26

4.5 THE DETERMINATION OF BICARBONATE AND CARBONATE.

Inorganic carbon in water is present as H2C03, HC03(-) and C03(2-). These 3 compounds are in equilibrium with each other according to;

H2C03 < > H( + ) + HC03(-)

HC03(-) < > H( + ) + C03(2-)

The two equilibrium constants of these equations are;

[H(+)] [HC03(-)] - 6.38 Kl = = 10 (at 25°C) (1)

[H2C03]

[H(+)] [C03(2-)] - 10.38 K2 = =-10 - (at 25°C) (2)

[HC03(-)]

From these equations the distribution of H2C03, HC03(-) and C03(2-) can be calculated as a function of the pH. The pH or acidity is defined as the negative logarithm of the H(+) concentration(pH = -log [H( + )]). Results of this calculations are given in fig.12 (Bolt 1978).

Fig.12 Distribution of H2C03;HC03(-) and C03(2-) as a function of pH

Above a pH of 8.3 bicarbonate and carbonate are present and below 8.3 H2C03 and HC03(-). For instance at a pH of 9.5, 87% of the inorganic carbon is present as HC03(-) and 13% as C03(2-).

27

F i g u r e 9 o r e q u a t i o n 2 can a l s o be u s e d t o c heck t h e a n a l y s i s . By t a k i n g l o g a r i t h m s of bo th s i d e s of e qua t i on (2) the fo l lowing r e l a t i o n i s ob ta ined:

pH = 10.38 + log [C03(2-)] / [HC03(-)] (3)

Example 1: What is the pH of a solution in which the [C03(2-)] is 0.5 raeq/1 and the [HC03(-)J is 2 meq/1?

Answer: In equation (3) [C03(2-)J is expressed in mol/1; so

[C03(2-)] = 0.0005/2

pH = 10.38 + log [C03(2-)]/[HC03(-)] = 10.38 + log 0.00025/0.002 = 10.38 + (-0.90) = 9.48

Example 2: Is the analysis of a water sample with the following results correct? [C03(2-)] = 0.4 meq/1, [HC03(-)] = 2.1 meq/1 and pH = 8.4 _

Answer: pH = 10.38 + log [Co3(2-)]/[HC03(-)] 8.4 = 10.38 + log [C03(2-j\][HC03(-)] log [C03(2-)]/[HC03(-)] = -1.98 [C03(2-)]/[HC03(-)] = 10~-1.98 = 0.01

The observed value for [C03(2-) ] / [HC03(-) ] was (0.4/2)/2.1 = 0.10. So the data are not correct.

The titration

Carbonate and bicarbonate can be titrated with HCl. First the carbonate is titrated.

C03(2-) + H+ -> HC03(-).

At a pH of 8.3 all the carbonate has been converted .into bicarbonate. At this pH more than 99% of the inorganiccarbon is present as HC03(-). The amount of equivalents of HCl equals the amount of mol of C03(2-). From 8.3 till 4.1 bicarbonate is titrated.

HC03(-) + H+ -> H2C03.

At pH 4.1 all the inorganic carbon is present as H2C03. The bicarbonate titrated originates from the bicarbonate already present in the original sample and the bicarbonate created by the titration of carbonate. The endpoint can be established with the pH-meter. Another possibility is using two indicators: 1-Phenolphtaleine for the point at pH 8.3. 2-The methyl-orange for pH 4.1.

28

Calculations

For all calculations the following symbols are used:

A = ml of HCl-solution used to reach pH 8.3 B = ml of HCl-solution used to reach pH 4.1 n = normality of HCl

Bicarbonate and carbonate in water samples.

For ground and surface water usually 50 ml samples are taken. The method of calculations is as follows:

[C032~] = 1000/50 * 2 * A * n = 4 0 * A * n (meq/1)

[HC03~] = 1000/50 * (B-2A) * n = 20 * (B - 2A) * n (meq/1)

1000/50 is a factor to have the results in meq./1-units, the factor 2 is for converting [C032-] from mmol to meq.

4.6 OXYGEN DEMAND.

4.6.1 Biological.

The b i o l o g i c a l oxygen demand (BOD) i s a measure for t he oxygen necessary to degrade organic matter by micro-organism. In the method t he wa ter i s a l l owed t o s t and for 5 days and t he oxygen concent ra t ion i s measured a t the beginning and a f t e r 5 days. The d i f fe rence i s the BOD. Because the maximum oxygen concent ra t ion in wa te r i s about 10 mg /1 , wa ter samples wi th h igh BOD-values need a d i l u t i o n . D i l u t i o n i s done wi th a i r s a t u r a t e d water conta in ing d i f f e ren t n u t r i e n t s . During the 5-days period the samples a r e s t o r e d in t he dark because o t h e rw i s e a l g a e may produce oxygen. The samples mos t l y o r i g i n a t e from d r a i n a g e -cana l s . These c ana l s conta in some micro-organism and seeding of t h e w a t e r w i t h m i c r o - o r g n n ir.m i s not nocnr . r .nry. Tf w a t e r sampl or, are d i r e c t l y taken from a d ra inage- tube they need seeding wxth micro-organism for i ns tance from the d r a inage -cana l . The method i s n o t v e r y c o m p l i c a t e d b u t n e e d s a v e r y a c c u r a t e a dmin i s t r a t i on .

29

For accurate measurement take the following remarks in mind:

Always do a blank determination to know the BOD of the reagent. This must be about zero and may be a little bit higher (< 1 mg /1 if the dilution-water is seeded with microorganism) . If the oxygen concentration after 5 days is smaller than 1 mg/1, a higher dilution was necessary. This should take

- If poisons or desinfectants (chlorine) are present the BOD will be low, because the micro-organism will not work. The tap-water in Cairo contains chlorine. Therefore never use fresh tap-water for making dilutions.

4.6.2 C h e m i c a l .

The chemical oxygen demand (COD) is a measure for the oxygen necessary to oxidize organic matter completely. The oxidation is on a chemical way by dichromate. Because the oxidation is complete, the COD is always higher than the BOD. The ratio COD/BOD can be almost one for water containing only sugar till hundred or more for water containing stable humic acids. The advantage of COD above BOD is that the determination of COD takes less time. In about 3 hours the result of the determination is known, whereas BOD takes more than 5 days. The principle of the method is that organic matter is oxidized by dichromate in a strongly acid medium with silver as a catalist.

Cr2072- + 14H30+ + 6e -> 2Cr3+ + 21H20 organic matter -> xC02 + yH20 + zNH4 + ne

The excess of dichromate is titrated with a solution containing Fe(II) with ferroin as indicator

6Fe2+ -> 6Fe3+ + 6e Cr2072- + 14H30+ + 6e -> 2Cr3+ + 21H20.

Chloride can also be oxidized by dichromate and will give a high COD value. For this oxidation can be corrected by adding mercury, which complexes the chloride.

30

5. WORKING PROCEDURES OF INSTRUMENTS

5.1 ATOMIC ABSORPTION SPECTROPHOTOMETER.

5.1.1 G e n e r a l .

Start position

-fuel locked

-air switch off -SLIT normal -SIGNAL lamp -MODE cont -RECORDER abs -GAIN fully counterclockwise -LAMP fully counterclockwise ~BG corrector AA

ADJUSTMENT OF THE INSTRUMENT.

-Be sure that there is water in the loop of the draintube and at least 13 cm of water in the waste vessel.

-Plug in the compressor.

-Place the lamp you need in the lampholder. The black knobs have to be between the mica spacers in the lamp.

-Plug in the power cord of the lamp.

-Turn on the power.

-Turn SIGNAL control to lamp. Adjust the lamp current to the desired value (see table 5). Read the value on the lamp/energy meter. Never exceed the desired value.

-Turn the SIGNAL control to Setup.

-Set the SLIT to the proper value (see table 5).

-Set the wavelength to the proper value (see table 5).

-Turn the GAIN control until a reading on the lamp/energy meter of about 25 is obtained.

^Optimize the wavelength setting with fine to have a maximum signal on the lamp/energy meter. If the signal becomes more than 50,decrease it to 25 with GAIN control.

31

-Align the lamp with the two black knobs to obtain a maximum value in the lamp/energy meter. Close the lamp compartment door.

-Adjust the horizontal position of the burner (left knob) on 8.0 The horizontal position (right knob) on 7.

-Open the acetylene cylinder and set the pressure on 0.85 atrn.

-Set the air switch to air and control if the pressure on the reducer is 2.5 atm. Adjust the flow to 50.

-Open the fuel valve and ignite the flame.

-Check if the pressure of the acetylene is still 0.85 atm and set the flow on 25.

-Turn SIGNAL to ABS.

-Aspirate destilled water and press AZ.

-Aspirate the alignement solution (table 5).

-Adjust horizontal,vertical and turning of the burnerhead with the three knobs to obtain maximum absorbance. Read the absorbance on the digital display. The vertical position must be smaller than 7. Otherwise the burnerhead will block the light beam.

-Adjust the acetylene flow to obtain maximum absorbance (not more than 35)

-ONLY FOR MAGNESIUM. Turn the burnerhead on such a way that the alignement solution gives a reading of about 0.8.

CALIBRATION

-Set SIGNAL control to Cone.

-Set MODE control to Hold

-Enter an integration time of 3 seconds by pressing 3 and t.

-Aspirate the blank solution. Wait about 3 seconds and press the READ key. Continue to aspirate until the indicator light goes off. Press the AZ key.

-Aspirate the first standard solution (SI) and press READ. When the reading is obtained, enter the concentration of the standard on the keyboard and press SI.

32

-Aspirate the second standard solution (S2) and press READ. When the reading is obtained, enter the concentration of the standard on the keyboard and press S2

-Now you can measure your samples. Aspirate the sample and press READ. Note the value on the report.

-After every 5 samples measure the blank and a standard solution. If the values are changed,control if there is a soil particle in the sample tube. Remove this and repeat the calibration. Also repeat the last 5 samples.

TURNING OFF THE INSTRUMENT

-Aspirate after the last sample during 2 minutes destilled water.

-Close the fuel shut off.

-Set the air switch on off.

"Set all knobs in start position.

-Turn off the power of the instrument.

-Close the acetylene bottle. "•Turn off the compressor.

-After 30 minutes cover the instrument with the plastic cover.

33

Table (5) Values of different elements for the Perkin Elmer 373 atomic absorbtion spectrophotometer.

For other elements,see manual of the instrument.

element

Ca

Mg

Fe

Mn

Cu

Zn

Pb

Co

Cr

Na

K

wave length (nm)

422.7

285.2

248.3

279.5

324.7

213.9

283.3

240.7

357.9

589.6

769.9

slit

(nm)

0.7

0.7

0.2

0.2

0.7

0.7

0.7

0.2

0.7

0.7

2.0

lamp current (mA)

15

10

30

20

15

15

10

30

25

8

12

alignement solution (mg/ )

10

5

10

5

10

2

10

5

5

2

2

SI

(mg/l)

2

2

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

S2

(mg/l)

10

5

2

2

2

2

2

2

2

2

2

5.1.2 Heavy metals.

The heavy metal concentration in water is mostly very low. Therefore special care must be taken. It is necessary to clean the sample bottles with diluted nitric acid. After sampling, the samples must be acidified with nitric acid (1 ml to 100 ml sample). In practice this means that a sub-sample must be taken. To measure, the normal procedure of atomic absorption can be used. But take instead of 3 seconds integration time 10 seconds. Adjust also the blank between every 2 samples.

It is also possible to use the recorder. After optimizing the instrument, adjust;

SIGNAL ABS MODE CONT RECORDER TC3

34

Adjust the zero of the recorder to give a deflection of 10. Aspirate the highest standard and adjust the sensitivity of the recorder to give a reading between 50 and 100 (sensitivity 10 mV °r less). Measure the samples, the standards and between all the blank solution (see figure 13). The readings are obtained from the recorder paper and the differences between the blank and the sample.

)blank

É sample 3

blank

(blank sample 2

sample 1

folank

standard 2

blank

standard 1

blank

Figure 13 Example of recorder output with measurement of heavy metals (AAS).

35

5.1.3 Measurement of back ground.

-Background must be measured with all heavy metals. Measure it immediate after measuring the element.

-After measuring a certain element,leave everything as it was. Also the burner.

-Set SIGNAL to CONT.

-LAMP fully counterclockwise.

-Place the H2-lamp.

-Adjust the LAMP current to obtain 15 mA.

-Adjust the lamp with the two black knobs to obtain minimum absorbance.

-Aspirate the blank solution and press AZ. If 0.000 cannot be reached or if the reading is very" unstable,increasing of the lamp current can give better results (current not more than 25 mA) .

-Measure the standards and the samples by pressing READ.

-The obtained values must be substracted from the readings measured with the element lamp. For the standard solutions a reading of about zero has to be obtained.

5.1.4 Use of diluter.

For atomic absorp t ion t e s t tubes are used, which can con ta in 10 ml. This r e s t r i c t s the d i l u t i o n to be made in one time. The maximum d i lu t ion factor i s 20 times. To obtain high d i l u t i ons several steps are necessary. I t i s important to mix in between. For making standards use the following t ab l e .

36

Table 6 Standards prepared from stock solutions containing 1000 mg/l.

Concentration mg/1 dilution factor steps of dilution

0.5 2000 10 * 10 * 20

1.0 1000 10 * 10 * 10

2.0 500 10 * 10 * 5

3.0 333 20 * 16.6

4.0 250 20 * 12.5

5.0 200 20 * 10

7.5 133 20 * 6,67

10.0 100 10 * 10

15.0 66.7 10 * 6.67

20.0 50 10 * 5

25.0 40 10 * 4

50.0 20 20

Table 7 Position diluter with different dilutions.

Dilution factor Position left Position right

4 2.0 3.00

5 2.0 4.00

6.67 1.5 4.25

10 1.0 4.50

12.5 0.5 2.88

16.6 0.5 3.90

20 0.5 4.75

37

5.1.5 Use of autosampler.

-Calibrate on the normal way.

-Connect the recorder and adjust the zero of the recorder on 10.

-MODE to CONT.

-RECORDER t o TC3.

- A s p i r a t e t h e h i g h e s t sample and a d j u s t t h e s e n s i t i v i t y of the r e c o r d e r t o h a v e a r e a d i n g b e tween 50 and 100 . R e co r d e r v e l o c i t y on 1 cm/min.

-Connect t h e a u t o s amp l e r .

- P l a c e s t a n d a r d s , b l a n k s and samples in t h e a u t o s amp l e r . Af te r e v e r y 5 samples t h e r e must be a s t a nda r d .

- P l a c e t h e s topmarker a f t e r t h e l a s t s ample .

-Be s u r e d e s t i l l e d wa te r i s f lowing th rough t he washing p a r t .

-Sample and washing c y c l e bo th on 30 seconds

- s t a r t t h e sampler and t he r e c o r d e r .

- W r i t e d i r e c t l y a f t e r a n a l y s i s t h e name of t h e s a m p l e s on, t h e r e c o r d e r p ape r .

5 .2 USE OF THE DILUTER.

With t h e d i l u t e r i t i s p o s s i b l e t o make d i l u t i o n s up t o 40 t i m e s . The amount of s amp l e t a k e n by t h e i n s t r u m e n t can v a r y from 0.5 t o 5 ml . Th i s can be d i l u t e d w i th 0 t o 20 ml d i l u t i o n s o l u t i o n . To make a d i l u t i o n i t i s n e c e s s a r y t o c hoo s e t h e amount of s ample . Then i t i s p o s s i b l e t o c a l c u l a t e t he amount of d i l u t i o n s o l u t i o n .

Fo r exa rop l e : The d i l u t i o n n e c e s s a r y i s 6 t i m e s . The c h o i c e of s amp l e vo lume i s 2 m l . The amount of d i l u t i o n s o l u t i o n needed i s ;

(n - 1) * v

n = dilution factor v = volume of sample

In this case (6 - 1) * 2 = 10 ml, 10 ml is adjusted with the right knob on 5.00 The instrument multiplies this value with 2.

38

It is also possible to chose the end volume. This is for instance done with the sulphate determination. The sample volume can still be chosed, but has to be completed with an amount of dilution solution to obtain the desired volume. For example;

Desired volume 20 ml. Sample volume 4 ml. This must be completed with 16 ml. The dilution factor is; (16 + 4)/4 = 5 times.

5.3 SPECTROPHOTOMETER SPECTRONIC 21

How to use ?

-Turn on power.

-Lamp and mirror to VIS

-Set correct wavelength.

-Switch on absorbance.

-Insert a blank solution,

-Set the instrument with the thumbwheel on the bottom of the instrument on 0.000.

-If 0.000 cannot be reached use a higher sensitivity. Use the lowest sensitivity possible.

-Take the samples and read the absorbance.

-Control the blank (0.000) every 5 samples or 5 minutes.

5.4 OXYGEN-ELECTRODE.

Calibration.

-Connect the electrode to the A-SENCE and REF jacks on the back of the meter. Connect the shorting trap across B-SENCE and REF.

-Turn the mode selector switch to pH. Set the STD-value switches to 7.00 and the SLOPE switches to +59.16.

-Set the electrode function switch to OFF. Press the SET CONCN button on the meter. The display should read 7.000. If not you must have a look to the manual of the meter.

39

- T u r n t h e mode s w i t c h on t h e e l e c t r o d e t o BTCK. Good b a t t e r y o p e r a t i o n i s i n d i c a t e d by a r e a d i n g o f 1 3 . 00 o r more on t h e m e t e r .

- T u r n t h e mode s w i t c h on t h e e l e c t r o d e t o ZERO. Use t h e z e r o c a l i b r a t i o n c o n t r o l t o r e a d 0.00 on t h e m e t e r .

- I f n o t d o n e a l r e a d y , i n s e r t t h e e l e c t r o d e i n t o a BOD b o t t l e c o n t a i n i n g e n o u g h w a t e r t o j u s t c o v e r t h e b o t t o m . Make s u r e t h a t t h e e l e c t r o d e t i p i s n o t immersed i n t o t h e w a t e r and d oe s n o t h a v e w a t e r d r o p l e t s c l i n g i n g t o t h e o u t s i d e o f t h e m e m b r a n e . Use t h i s b o t t l e a l s o f o r s t o r a g e o f t h e e l e c t r o d e b e t w e e n m e a s u r e m e n t s .

- T u r n t h e e l e c t r o d e mode s w i t c h t o t h e AIR p o s i t i o n . Use t h e AIR c a l i b r a t i o n c o n t r o l t o r e a d 7.600 on t h e m e t e r . 7.600 i s t h e b a r o m e t e r p r e s s u r e (mm Hg) d i v i d e d by 100 .

- T u r n t h e mode s w i t c h on t h e e l e c t r o d e t o H20 a nd m e a s u r e t h e s a m p l e s .

- A f t e r m e a s u r i n g t u r n t h e e l e c t r o d e mode s w i t c h t o OFF.

5 . 5 CALIBRATION OF SARTORIUS 2002 MP 1 BALANCE.

1 - C l e a n b a l a n c e pan and c l o s e s l i d i n g d o o r s .

2 - Remove t h e s m a l l b l a c k c o v e r a t t h e r i g h t of t h e i n s t r u m e n t .

3 - P r e s s t h e r e d b u t t o n t o c a l l up t h e c a l i b r a t i o n p r o g r a m .

4 - When t h e r e a d i n g i s s t a b l e , p r e s s [TARA],

5 - P l a c e a w e i g h t of 1 5 . 0 00 g . on t h e b a l a n c e .

6 - When t h e r e a d i n g i s s t a b l e , p r e s s [TARA].

7 - R e l e a s e t h e r e d s w i t c h , by p r e s s i n g i t a g a i n .

8 - C l o s e w i t h t h e b l a c k c o v e r .

40

6 . C H E M I C A L S A N D S O L U T I O N S

6 . 1 PREPARATION OF TITRATION SOLUTIONS,

Water s amples

0.05 n AgN03: D i s s o l v e 8 .4935 g AgN03 i n 1 l i t e r of d i s t i l l e d wa te r (measuring f l a s k ) .

0.1 nHCl : D i l u t e 8.3 ml of c o n c e n t r a t e d HCl t i l l 1 l i t e r w i th d i s t i l l e d water (measuring f l a s k ) .

0 . 0 1 m EDTA: D i s s o l v e 3.72 g Na2EDTA. 2H20 i n 1 l i t e r of d i s t i l l e d wa te r (measuring f l a s k ) .

S o i l - p a s t samples

0 .005 nAgN03:Disso l ve 0.8494 g AgN03 i n 1 l i t e r of d i s t i l l e d wa te r (measuring f l a s k ) .

0.01 n HCl: D i l u t e 0.83 ml of c o n c e n t r a t e d HCl t i l l 1 l i t e r w i th d i s t i l l e d wa te r (measuring f l a s k ) .

O.OlmEDTA: D i s s o l v e 3.72 g Na2EDTA. 2H20 i n 1 l i t e r of d i s t i l l e d wa te r (measuring f l a s k ) .

6 . 2 STANDARD SOLUTIONS

0.05 n NaCl: D i s s o l v e 2 .9225 g NaCl i n 1 l i t e r of d i s t i l l e d wa te r (measuring f l a s k ) . The NaCL must be d r i e d du r ing 2 hours a t 300 C.

0 . 0 1 raCaCL2: Weight 1.000 g CaC03. Pu t t h i s i n a m e a s u r i n g f l a s k of 1 l i t e r . Add 50 ml 1 n HCl. D i l u t e t i l l 1 l i t e r w i t h d i s t i l l e d w a t e r . The CaC03 must be d r i e d du r ing 2 hours a t 400 C.

0.05 n N a2C03 :D i s so lve 2.6500 g Na2C03 in 1 l i t e r of d i s t i l l e d wa te r (measuring f l a s k ) . The Na2C03 must be d r i e d du r ing 4 hours a t 250 C.

41

6.3 ADJUSTING NORMALITIES.

AgN03

0.005n: P i p e t 1 ml of 0.05 n NaCL s t a nda r d s o l u t i o n . T rea t t h i s as a normal sample. Note the amount necessary to reach the endpoint .

The normali ty i s ; 0.05

Amount titrated (ml)

0.05 n: Pipet 10 ml of 0.05 n NaCL standard solution. Treat this as a normal sample. Note the amount necessary to reach the endpoint.

The normality is; 0.5

Amount t i t r a t e d (ml)

HCl

0 .01 n : P i p e t 2 ml of 0.05 n Na2C03 s t a nd a r d s o l u t i o n . T rea t t h i s as a normal sample. Note the amount necessary to reach the endpoint .

The normality i s ; 0.1


or T i t r a t e 20.00 ml HCL in a beaker of 100 m l .T i t r a t e t h i s s o l u t i o n w i t h 0.05 n AgN03 t o measure t he c h l o r i d e . Note t h e volume n e ce s s a ry t o r each t he endpo in t .

The normal i ty i s ;

Volume AgN03 X normality AgN03

20.00

42

0 .1 n : P i p e t 20 ml of 0.05 n Na2C03 S t a n d a r d s o l u t i o n . T r e a t t h i s as a normal s ample . Note t h e amount n eces sa ry t o r each t h e e ndpo in t .

The no rma l i t y i s ; 1.0


o r T i t r a t e 5.00 ml HCL i n a b e a k e r of 100 m l . T i t r a t e t h i s s o l u t i o n w i t h 0 .05 n AgN03 t o m e a s u r e t h e c h l o r i d e . Note t h e vo lume n e c e s s a r y t o r e a c h t h e e n d p o i n t .

The no rma l i t y i s ;

Volume AgN03 X no rma l i t y AgN03

5.00

Ferrous ammonium sulphate solution: see also 6.15

Pipet 10.0 ml standard dichromate solution 0.25 N and dilute to about 250 ml. Add 20 ml cone. H2S04 and allow to cool. Titrate against the ferrous ammonium sulphate using 2 or 3 drops of ferroin indicator.

The normality is: 2.50

amount t i t r a t e d (ml)

EDTA

0 .01 M: P i p e t 10 ml of 0.01 M CaC12 in a b eake r of 100 ml . T r e a t t h i s a s a n o rma l s amp l e t o d e t e r m i n e c a l c i u m . Note t h e amount n e c e s s a r y t o r each t h e e ndpo in t . The m o l a r i t y i s ;

0.10

amount titrated (ml)

If in the buffer the magnesium salt of EDTA is used the molarity is the same for the determination of Ca and Ca + Mg. If another magnesium salt is used, the molarity has to be established for both titrations.

43

6.4 pH

P r e p a r e b u f f e r s o l u t i o n s f rom s t a n d a r d t a b l e t s o r s o l u t i o n s . P r epa re f r e sh s o l u t i o n s eve ry month and compare new s o l u t i o n s w i th o l d ones .

6 . 5 CONDUCTIVITY

0.05 m KCl: D i s s o l v e 3.725 g KCl in 1 l i t e r of d i s t i l l e d water (measuring f l a s k )

0.01 m KCl: D i s s o l v e 0 .745 g KCl i n 1 l i t e r of d i s t i l l e d w a t e r (measuring f l a s k )

6 . 6 CHLORIDE.

-Indicator potassiumchromate: dissolve 5 g potassiumchromate in 50 ml distilled water.

-1 N HN03: dilute 7 ml cone. HN03 to 1 liter.

6.7 BICARBONATE.

-Phenolphtalein indicator: dissolve 1 g phenolphtalein in 100 ml ethanol 96%.

-Methylorange indicator: dissolve 100 mg methylorange in 1 liter distilled water.

6.8 CALCIUM AND MAGNESIUM.

-Buffer solution: dissolve 16.9 g ammoniumchloride (NH4C1) in 143 ml cone, ammoniumhydroxide (NH40H). Add 1.25 g magnesium salt of EDTA (MgEDTA) and dilute to 250 ml with distilled water.

-Indicator solution: dissolve 0.5 g eriochrome-black-T and 4.5 g of hydroxylaminehydrochloride in 100 ml of ethanol 96%.

-Lanthaniumchloride solution for use with AAS: dissolve 115 g LaC13 in 1 liter 1 mol/1 HCl. SrCl2 can also be used (180 g instead of 115 g lanthaniumchloride). The measuring solutions have to contain 5 ml LaCl3-solution in 100 ml.

44

6.9 CALCIUM.

-4 M NaOH: dissolve 160 g of NaOH in 1 liter distilled water.

-Indicator ammoniumpurpurate : thoroughly mix 0.5 g of ammoniumpurpurate with 100 g of powdered potassiumsulphate.

6.10 SULPHATE

- Conditioning reagent Dissolve in 800 ml distilled water: 75 g NaCl. Add: 30 ml concentrated hydrochloric acid,100 ml 95 % ethanol and 50 ml glycerine (glycerol) mix well.

- Bariumchloride crystals.

- S t anda rd s u l p h a t e s o l u t i o n 1000mg/l Weight 1.479 Na2S04 and d i s s o l v e in 1 l i t e r wa ter (measuring f l a s k ) . The Na2S04 must be d r i e d a t 300 C for 4 h ou r s . P i p e t from t h i s s o l u t i o n 1 ; 2 ; 3 ; 4 ; 5 ; 6 ; 7 ; 8 ; 9 and 10 ml in measuring f l a s k s of 100 ml. This g i v e s s o l u t i o n s of 10 ; 20 ; 30 ; 40 ; 50 ; 60 ; 70 ;80 ; 90 and 100 mg/1.

6 . 1 1 SODIUM and POTASSIUM.

-0.1 M NaCl d i s s o l v e 5.844 g NaCl in 1 l i t e r of d i s t i l l e d water (measuring f l a s k ) .

- 0 . 1 M KCL d i sso lve 7.455 g KCL in 1 l i t e r of d i s t i l l e d water .

6 . 1 2 NITRATE.

-Sulphuric acid 18 mol/L (p=1.84 g/L)

-Sodiumhydroxide 10 mol/L. Dissolve carefully 400 g NaOH in 600 ml water. Dilute till 1000 ml.

-Sodiumsalicylate solution. Dissolve 0.5 g sodiumsalicylate in 100 ml water. This solution must be prepared daily. Stock nitrate solution 0.1 mg/ml N03-N. Dry potassiumnitrate KN03 for 1 hourat 120°C. Weight 722 mg and dissolve in water in a measuring flask (1000 ml).

45

-Standard nitrate solution. Dilute 20 ml of the stock-solution (see 2.4) till 100 ml in a measuring flask. This solution contains 0.02 mg/ml N03-N.

-Standard nitrite solution 3 ug/ml N02-N. Dilute 20.0 ml of the stocksolution (2.5) to 1000 ml in a measuring flask and mix.

6.13 NITRITE.

Use o n l y r e a g e n t s o f P.A. q u a l i t y a n d d é m i n e r a 1 i z e d w a t e r t h a t i s a l s o d e s t i l l e d .

- H y d r o c h l o r i c a c i d (HCl) 6 m o l / L .

-Na2 EDTA- s o l u t i o n 0 . 025 m o l / L . D i s s o l v e 10 g d i s o d i u m -e t h y l e n e - d i a m i n e - t e t r a a c e t a t e , Na2-EDTA.-2H20 i n w a t e r , d i l u t e t i l l 1 l i t e r and mix . S t o r e t h i s s o l u t i o n i n a p o l y e t h y l e n e b o t t l e .

- S u 1 f a n i 1 a m i d s o l u t i o n . D i s s o l v e 2 00 mg 4 - a m i n o b e n z e n e s u l f o n i c a c i d amide ( s u l f o n a m i d e ) i n 200 ml w a t e r .

- N - ( l - n a f t y l ) 1.2 - d i a m i n o - e t h a n e - h y d r o c h l o r i d e s o l u t i o n -D i s s o l v e 100 mg of t h i s r e a g e n t i n 100 ml w a t e r .

S t o c k n i t r i t e s o l u t i o n , N02 -N c o n t e n t s 150 m g / L . Dry s o d i u m n i t r i t e , NaN02, d u r i n g 1 h a t 110 C. D i s s o l v e 740 mg i n 1 l i t e r w a t e r .

REMARK.

Sodiumnitrite solutions are not stable. The tenability cat1

be improved by use of sterilized bottles, fresh boiled water an° adding 1 ml chloroform to one liter. This solution can stored for one day.

6.14 BIOLOGICAL OXYGEN DEMAND.

-Phosphate buffer: dissolve 8.5 g KH2P04; 21.75 g K2HP04; 33.4 0 Na2HP04.7H20 and 1.7 g NH4C1 in about 500 ml distilled wate* and dilute to 1 liter. The pH of this buffer should be 7.2.

-Magnesiumsu lphate solution: dissolve 22.5 g MgS04.7H20 i*1

distilled water and dilute to 1 liter.

-Calciumchloride solution: dissolve 27.3 g anhydrous CaC12 ^ distilled water and dilute till 1 liter.

46

•Fe r r i ch lo r ide s o l u t i on : d i s s o l v e 0.25 g FeC13.6H20 in d i s t i l l e d water and d i l u t e t i l l 1 l i t e r .

-Di lut ion s o l u t i on : prepare fresh before use. P lace the des i red volume of d i s t i l l e d water in a s u i t a b l e b o t t l e and add 1 ml each of phospha te b u f f e r , magnesiumsulphate , ca lc iumchlor ide and f e r r i c c h l o r i d e s o l u t i on s for each l i t e r of water. Aerate t h i s s o l u t i on for 2 hours.

6 . 1 5 CHEMICAL OXYGEN DEMAND,

-Standard po t a s s iumdich roma te s o l u t i o n 0.250 N: d i s s o l v e 12.259 g K2Cr207 d r i e d a t 105°C f o r 2 h o u r s i n d i s t i l l e d w a t e r and d i l u t e t o 1000 ml (measuring f l a s k ) .

-Cone. H2S04 w i t h s i l v e r : add 10 g Ag2S04 t o 1 l i t e r c o n e . H2S04. Leave i t f o r a day t o d i s s o l v e .

-S tandard fe r rousammoniumsulphate s o l u t i o n 0.25 N: d i s s o l v e 98 g Fe(NH4)2(S04)2.6H20 in d i s t i l l e d wa t e r . Add 20 ml cone . H2S04, c o o l and d i l u t e t o 1000 ml (mea su r i ng f l a s k ) . T h i s s o l u t i o n must be s t a n d a r d i z e d a g a i n s t t he K2Cr207 d a i l y ( see 6.3).

- F e r r o i n i n d i c a t o r : t h i s i n d i c a t o r s o l u t i o n can be p u r c h a s e d a l r e a d y p r e p a r ed (Merck).

47

7. DESCRIPTION OF METHODS USED

7.1 pH

Calibration of pH-meters

7.1.1 pH-raeter SCHOTT CG820

-Rinse the electrode with distilled water.

-Take buffer pH-7.

-Adjust the meter with pH to read 7.


-Take buffer pH-4 and adjust the meter to read 4 with mV/pH.


-Control the meter with buffer pH-7. If the reading is 7.0 ,the calibration was correct. If the deviation is smaller than 0.1 unit re-adjust and the calibration is also correct. If the deviation is more than 0.1 unit,start again.

-Change the reading of the instrument to mV and measure the potential of buffer pH-7. Note this value in the control table.

-Read the value of mV/pH and note in the control table. If this is not possible,measure also the potential of buffer pH-4 and note this value in the control table.

-Change the reading of the instrument to pH and start measurements.

7.1.2 pH-raeter ORION model 601

-The back most knob (right of panel) must be on 100% The front black knob on the temperature of the buffers.


-Take buffer pH-7.

-Adjust the meter with calib to read 7.


48

-Take buffer pH-4 and adjust the meter to read 4 with the front black knob.

-Adjust the back most knob to give the temperature of the buffers. Do not touch the front most knob.


-Control the meter with buffer pH-7. If the reading is 7.0, the calibration was correct. If the deviation is smaller than 0.1 unit re-adjust and the calibration is also correct. If the deviation is more than 0.1 unit, start again.

-Change the reading of the instrument to mV and measure the potential of buffer pH-7. Note this value in the control table.

-Read the value of % slope and note in the control table. If this is not possible, measure also the potential of buffer pH-4 and note this value in the control table.

-Change the reading of the instrument to pH and start measurements.

table 8; pH control table. Slope is reading from the meter or(mV pH-7 minus mV pH-4) divided by 3

name date mV of pH-7 mV of pH-4 slope remarks

Joop 26/11/88 -19 155 -58.0

7.1.3 Check of the meters.

On the laboratory two instruments must be ready to use. Each pH-meter with its own buffers. Measure every two weeks 5 samples on both meters. The average difference must be smaller than 0.1 unit. If not re-calibrate or make new buffer solutions.

49

10 r

5 °\-e T -10

-20

-30

/l Meter 1 59

• i • . 58

o - W ' 1 / - ° ' \ / W - - . ' ' v \

57

56 _1 I 1 I 1 1 1 I 1 1 I I — 5 5

^V/V >'\ /°-c-*°'°-°'°^-<>'°-—°

J I I I L_J I I I I I I

10 r

> E

0 -

-10

Meter 2 " ^ ' • ' ^ \ / v \ '«-»-«.-•'.-»-».„.o

^ 5 9 r

58

57

56 K V V J I I I I l I I I I I I 55

/"% / " WS-"*"—-•"•'*"•-

_ 1 I I I I I I I I I — I I J F M A M J J A S O N D

Time

0 2

01 -

<n 0

x 1 - 0 1

- \„^-N>'-j i i 3 i i I _ I i i i

J F M A M J J A S O N D Time

f igure IA Example of con t ro l c a r t s for the pH-meters

In t h e example, t h e d i f f e r e n c e in t h e r e s u l t s of t h e 2 pH-m e t e r s i s t o o h i g h i n J u n e . T h i s i s c a u s e d by a wrong c a l i b r a t i o n of meter 1. After c o r r ec t i on of the c a l i b r a t i o n , the r e s u l t s a r e t h e same a g a i n . As a r e s u l t t he s amples measured between t h e wrong c a l i b r a t i o n and the r e - c a l i b r a t i o n need a c o r r e c t i on of 0.2 pH un i t .

50

7.2 CONDUCTIVITY

Calibration

7.2.1 Analytical Control Instrument.

-Be sure that on the right of the instrument only the lamp of 3 kHz is on.

-Take the 0.0 5 m KCl solution

-Measure the temperature of the solution. Adjust the temperature on the meter.

-Rinse the cell with some KCl solution.

-Measure the conductivity. Adjust with CONST ADJ to read 6.66 .

-Note the value of CONST ADJ in the control table.

-Measure the conductivity of the 0.01 m KCl solution. The reading must be 1.41 + 0.05. If not, make new solutions. Note the value in the control table.

7.2.2 PTI 18 instrument

-Take the 0.05 m KCl solution.

-Measure the temperature of the solution.

-Rinse the cell with some KCl solution.

-Measure the conductivity. Adjust with CELL to read the value presented in table 8.

-Measure the conductivity of the 0.01 m KCl solution. The reading must be the value given in table 8 +0.05. If not,make new solutions. Note the value in the control table.

51

Table 8 Specific conductance of KCl solutions (mS/cm=mho/cm) as a function of temperature (C)

temperature 0.01 m KCl 0.05 m KCl

15 1.147 5.404

16 1.173 5.527 17 1.199 5.651 18 1.225 5.775 19 1.251 5.889 20 1.278 6.024

21 1.305 6.149 22 1.332 6.275 23 1.359 6.402 24 1.386 6.529 25 1.413 6.656

26 1.440 6.784 27 1.468 6.912 28 1.496 7.041 29 1.524 7.170 30 1.552 7.300

31 1.580 7.430 32 1.609 7.561 33 1.637 7.692 34 1.666 7.824 35 1.695 7.956

Control table for Analytical Control instrument

name date const adj 0.01 m KCl remarks

Joop 6/4/87 1.10 1.39 new inst, new sol.

52

7.2.3 Measurement of samples.

Analytical Control instrument

-Be sure that on the right of the instrument only the lamp of 3 kHz is on.

-Rinse the electrode.

-Measure the temperature of the samples.

-Adjust this value on the instrument with TEMP COMP.

-Measure the conductivity of the samples. Note the values and also the name of the instrument. There is no need to correct the values for temperature. This is done by the instrument.

PTI 18 instrument.

-Rinse the electrode.

-Measure the temperature of the samples and note this on the report.

-Measure the conductivity of the samples. Note the values and also the name of the instrument.

-Correct the values for the temperature as follows: Conductivity (ms/cm 25 C) = factor x reading. The factor to correct for temperature is given in table 9.

Table 9 Temperature correctionfactors for conductiviy.

Temperature Factor Temperature Factor

15 16 17 18 19 20

21 22 23 24 25

1.246 1.211 1.189 1.161 1.135 1.110

1.084 1.063 1.041 1.020 1.000

26 27 28 29 30

0.979 0.958 0.938 0.919 0.899

53

If t h e s t a nd a r d s o l u t i o n s and t he samples have t h e same t empe r a t u r e i t i s p o s s i b l e t o measure on a n o n - c a l i b r a t e d i n s t r u m e n t . Measure t h e r e f o r e t h e c o n d u c t i v i t y of t h e KC1-s o l u t i o n s and t he s amp le s . Co r r ec t t h e measured v a l u e s on t he fo l lowing way;

f a c t o r l =6.656/measured value 0.05 KCl fac tor2 =1.413/measured value 0.01 KCl

If t h e 2 f a c t o r s d i f f e r not more then 3 %,take t he a ve r age and m u l t i p l y t h e measured v a l u e s of t h e samples wi th t h i s f a c to r . If not make new s o l u t i on s and check what i s wrong.

Use the a n a l y t i c a l c on t ro l instrument as a r e fe rence . Measure e ve ry month on t h i s meter and a l l o t h e r me te r s 5 s amp le s . C a l c u l a t e t h e r e l a t i v e d i f f e r e n c e (%) ( r e ad ing m e t e r / r e a d i n g re ference - l)xl00% take the average of the 5 samples. Note t h i s in a c o n t r o l t a b l e . The a b s o l u t e d i f f e r e n c e must be s m a l l e r than 3 %. If not , c l ean ing of the e l e c t r ode with HCl can improve the r e s u l t s .

7 .3 TITRATIONS

S o i l pa s t e s amp l e s .

C h l o r i d e , b i c a r b o n a t e , c a l c i um and magnesium a r e d e t e rm ina t ed w i t h a i d of t i t r a t i o n . The amount of sample and t h e t i t r a t i o n s o l u t i o n a r e g i v en i n t a b l e 10.

T ab l e lO : Summary of amount of sample and t i t r a t i o n s o l u t i o n fo r t i t r a t i o n methods.

S o i l p a s t e samples

Amount sample t i t r a t i o n s o l u t i o n

CL 2 ml 0 .005 n AgN03

HC03 Ca

Ca + Mg

5 ml 1 ml

1 ml

0 . 0 1 n HCl 0 . 0 1 n EDTA

0 . 0 1 n EDTA

54

Water samples

Amount sample titration solution

CL

HC03

Ca

Ca + Mg

20

50

10

10

ml

ml

ml

ml

0.0 5 n AgN03

0.1 n HCl

0.01 n EDTA

0.01 n EDTA

7.4 CHLORIDE.

7.4.1 Using electrode.

water samples

-Pipet 20 ml sample in a beaker of 100 ml.

-Add 1.25 ml 1 n HN03.

-Add a magnetic stirrer.

-Dilute to be 60 ml with distilled water.

-Titrate with 0.05 n AgN03 till a potential of -120 mV is read on the mV-meter.

-Note the amount of AgN03 used (a ml).

-Note the normality of the solution.

CALCULATION

n a

-[CI] = 50 x n x a

normality of AgN03 solution amount of AgN03 used for sample

(meq/1)

If another sample volume b igger / sma l l e r than 20 ml i s used, mu l t i p l y with; 20/sample volume (ml).

55

Soil paste samples


-Add 1.25 ml 1 n HN03.


-Dilute to be 60 ml with distilled water.

-Titrate with 0.005 n AgN03 till a potential of -120 mV is read on the mV-meter.



CALCULATION

-[CI] = 500 x n x a (meq/1)

n = normality of AgN03 solution a = amount of AgN03 used for sample

If another sample volume bigger/ smaller than 2.0 ml is used, multiply with; 2.0/sample volume (ml).

7.4.2 Using potassium Chromate as indicator.

water samples

-Pipet 20 ml sample in an erlenmeyer flask of 100 ml.

-Add 10 drops (about 0.2 ml) indicator solution.

-Titrate with 0.05 n AgN03 to a pinkish/yellow endpoint.


-Do a blank titration using 20 ml distilled water. -Note also this amount used (b ml).


56

CALCULATION

-[Cl] = 50 x n x (a - b) (meq/1)

n = normality of AgN03 solution a = amount of AgN03 used for sample b = amount of AgN03 used for blank

If another sample volume bigger/ smaller than 20 ml is used, multiply with; 20/sample volume (ml).

soil paste samples


-Add 5 drops (about 0.1 ml) indicator solution.

-Titrate with 0.005 n AgN03 to a pinkish/yellow endpoint.


-Do a blank titration using 2 ml distilled water.

-Note also this amount used (b ml).


CALCULATION

-[CI] = 500 x n x (a - b) (meq/1)

n = normality of AgN03 solution. a = amount of AgN03 used for sample.


57

7.5 BICARBONATE AND CARBONATE

7.5.1 Using glass - electrode.

water samples



-Measure the pH with a calibrated pH-meter and note the value on the report.

-If the pH is more than 8.3,titrate with 0.1 HCL till that value is reached .

-Note the amount of HCl used (a ml)

-Titrate with 0.1 n HCl till a pH of 4.1 is reached.

-Note also this amount used (b ml)


CALCULATION.

-[C03] = 40 x n x a (meq/1)

-[HCO3] =20 x n (b - 2a) (meq/1)

n = normality of HCl solution. a = amount of HCl used to reach pH 8.3 b = amount of HCL used to reach pH 4.1


Soil paste samples

With soil paste samples only 5 ml sample is taken. This is not enough for the electrode. Dilution of the sample gives a small change in the pH. This could lead to errors for samples with an orriginal pH of more than 8.3.

58

7.5.2 Using indicator solutions.

water samples


-Add 1 drop phenolphtaleine indicator solution.

-If the solution has a red color titrate with 0.1 n HCL till the color dissapears.

-Note the amount of HCl used (a ml).

-Add 2 drops methyl-orange indicator solution.

-Titrate with 0.1 n HCl till the color changes from yellow to orange.

-Note also this amount used (b ml).


CALCULATION.

-[C03] = 40 x n x a (meq/1)

-[HC03] =20 x n (b - 2a) (meq/1)

n = normality of HCl solution a = amount of HCl used for colorchange of phenolphtaleine. b = amount of HCL used for colorchange of methyl-orange.


Soil paste samples


-Add 1 drop phenolphtaleine indicator solution.

-If the solution has a red color titrate with 0.01 n HCL till the color dissapears.

-Note the amount of HCl used (a ml). -Add 2 drops methyl-orange indicator solution.

-Titrate with 0.01 n HCl till the color changes from yellow to orange.


59


CALCULATION.

-[C03] = 400 x n x a (meq/1)

-[HC03] =200 x n (b - 2a) (meq/1)

n = normality of HCl solution a = amount of HCl used for colorchange of phenolphtaleine. b = amount of HCL used for colorchange of methyl-orange.


7.6 CALCIUM AND MAGNESIUM

7.6.1 Water samples.


-Add 0.5 ml buffersolution (ammoniumch1oride-ammonium hydroxide buffer).

-Add 3 or 4 drops of Eriochrome black T indicator.

-Titrate with 0.01 m EDTA till the color changes from wine red to blue.

-Note the amount of EDTA used (b ml)

-Do a blank titration using 20 ml destilled water.



CALCULATION.

-[Ca + Mg] = 100 x m x b (meq/1)

m = normality of EDTA solution b = amount of EDTA used in ml.

If another sample volume bigger/ smaller than 10 ml is used, multiply with 10/sample volume (ml).

60

7.6.2 Soil paste samples.


-Dilute till about 10 ml with distilled water.

-Add 3 a 4 drops (about 0.1 ml) Eriochrome black T indicator

-Titrate with 0.01 m EDTA to a wine-red/blue endpoint.

-Note the amount of EDTA used ( b ml).

-Note the molarity of the solution.

CALCULATION.

-[Ca] =1000 x m x b (meq/1)

m = m o l a r i t y o f E D T A s o l u t i o n . b = amount of EDTA u s e d f o r s a m p l e .

I f a n o t h e r s a m p l e v o l ume b i g g e r / s m a l l e r t h a n 1.0 ml i s u s e d , m u l t i p l y w i t h ; 1 . 0 / s a m p l e v o l ume ( m l ) .

7 . 7 CALCIUM

7.7.1 Water samples.


-Add 5 drops (about 0.1 ml) sodium hydroxide 4 N.

-Add about 50 mg of ammonium purpurate indicator. -Titrate with 0.01 m EDTA till the color changes from orange-red

to purple.

-Note the amount of EDTA used (b ml).


61

CALCULATION.

-[Ca] = 100 x m x b (meq/1)

m = molarity of EDTAsolution. b = amount of EDTA used for sample.


7.7.2 Soil paste samples.


-Dilute till about 10 ml with distilled water.

-Add 5 drops (about 0.1 ml) sodium hydroxide 4 N solution.

-Titrate with 0.01 m EDTA to a orange-red/purple endpoint,

-Note the amount of EDTA used (b ml).


CALCULATION.

-[Ca] = 1000 x n x b (meq/1)

m = molarity of EDTA solution. b = amount of EDTA used for sample.


7.8 SULPHATE

Principle.

Sulphate is precipitated in hydrochloric acid medium with bariumchloride to form bariumsulphate. This is kept in

suspension with a conditioning reagent. The absorption of light by the suspension is measured with a spectrophotometer. The sulphate concentration is established by comparing of the reading with those of standard solutions (standard curve).

62

Ins t rumenta t ion .

- Spec t r opho tome t e r fo r use a t a wave l eng th of 420 nm. C e l l l ength 1 cm.

-Magnetic s t i r r e r .

-Stopwatch.

-Erlenmeyer flasks of 100 ml.

Reagents.

- Conditioning reagent.

- Bariumchloride crystals.

- Standard sulphate solution 1000mg/l.

Procedure.

This procedure is valid for the standard solutions as well as for the samples.

-Pipet 20 ml of the sample in a erlenmeyer flask of 100 ml.

-Add 2 ml of conditioning reagent.

-Add a magnetic rod and place the the flask on a stirrer.PLace a funnel on the flask.

-Add 0.10 g bariumchloride.

-Stir exactly during 1 minute.Adjust during this period the spectrophotometer on 0.000 using destilled water.

-Measure the absorbance on the spectrophotometer at 420 nm. Read within 4 minutes. Take the highest reading obtained during this period. If the absorbance is still getting higher, take the absorbance after 4 minutes.(this is 5 minutes after adding the bariumchloride). If the reading is more than the reading of the highest standard,dilute the sample (see dilution table).

-Measure a blank, using 20 ml of the sample»adding all the reagents except bariumchloride. Read the absorbance at 420 nm. Depending on the color of the sample the reading will be 0.000 or more.

63

Calculation.

Calculate the absorbance caused by sulphate;

A = As - Ab

A = Absorption caused by sulphate. As= Absorption of sample with BaS04. Ab= absorption without BaS04 (blank).

Make a calibration curve as shown (fig.15 ). Read the amount of sulphate from the curve. Multiply it with the dilution used.

Dilutions

For the sulphate determination it is possible to use the diluter. The end volume has to be 20 ml. This makes the following dilutions possible.

V 5 r

10

S 0 5 <

00

: -_ -

--

-/

** s' I 1 1

/ / •

1

s*

. / •

y .r /

i i i i i i 20 40 60 80 100 120

Concentration (ppm)

Figure 15 Calibration curve of sulphate,

Table 10 Dilution table for the sulphate determination using the diluter.

Dilution factor Knob left Knob right

5 10 20 40

4.0 2.0 1.0 0.5

8.00 9.00 9.50 9.75

64

7.9 SODIUM AND POTASSIUM.

Calibration of the meter (monthly)

- Prepare standard solutions with 0.5; 1.0; 1.5; 2; 3;4 and 5 meq Na( + ) and K( + ) per liter. For this purpose, dilute 5; 10; 15; 20; 30; 40 and 50 ml of the 0.1 M NaCl + KCl solution to 1 liter with distilled water.

- Turn on the meter and light the flame. The flame has to be blue.

- Install the sodium or potassium filter.

- Allow a warm-up period

- Aspirate alternately distilled water and the 5 meq/1 solution and operate the sensitivity controls untill the reading of the distilled water is zero and the reading of the standard solution is 100.

- Aspirate all the standard solution and note the readings.

- Make a calibration curve. X-axis = concentration, Y-axis = reading.

Measurements,

- Use the standard solutions with 2 and 5 meq/1.

- Turn on the meter and light the flame. The flame has to be blue.

- Install the sodium or potassium filter.

- Allow a warm-up period of 10 minutes.

- Aspirate alternately distilled water and the 5 meq/1 solution and operate the sensitivity controls untill the reading of the distilled water is zero and the reading of the standard solution is 100.

- Aspirate the 2 meq/1 solution. Compare the reading of the standard with the value in the calibration curve. A deviation of 3 % is allowed. If the deviation is more then 3 %, check the solutions used. If no mistake is found, prepare new solutions. If necessary a new calibration-curve.

- Aspirate the samples. Note the reading. If the reading is more then 100, dilute the sample 10 times. Note the dilution which is made.

65

- Check the performance of the photometer every 5 samples by aspirating the blank and the 5 meq/1 solution. Adjust the sensitivity as necessary. If the reading of the highest standard has changed considerable inspect the nebuliser and tubing for clogging and clean if necessary.

Calculation.

- Read the concentration from the calibration curve. It is also allowed to use a mathematical function describing this curve.

- Multiply the value from this curve with the dilution factor.

- If only low concentration < 1 meq/1 are measured, make a calibration curve in this concentration region. An advantage of this low range is, that the calibration curve is almost linear.

- If a mathematical description of the calibration curve is used, be sure that this function is also changed when the calibration curve is changed.

7.10 PHOTOMETRIC DETERMINATION OF NITRATE.

Salicylic acid is nitrated in a sulphuric acid medium by nitrate. The produced nitrocompound has an intensive yellow colour in a alkalic medium. The absorbance at 415 nm is a measure for the amount of nitrate.

Apparatus.

A spectrophotometer suitable for measurements at 415 nm. Measering cells with a pathway of 10 mm.

Sample.

Samples for n i t r a t e can be s t o r e d on a c oo l p l a c e (4 C) for one week. If b i o l o g i c a l a c t i v i t y w i l l occur the sample must be a c i d i f i ed with H2S04 t i l l pH 2.

66

Procedure.

- P i p e t v ml of sample c o n t a i n i n g not more than 0.5 mg N03 in a beaker of 50 ml.

- Add 1 drop of p heno lph t a l e i n e and add as much 0.1 M NaOH t i l l the red co lour appears.

- Add 2 ml of sodiumsal icylaa t r eagen t .

- Dry on a water bath (100 C) t i l l the sample i s completely dry.

- Add 2 ml of s u l p h u r i c a c id (18 mol/L) and be s u r e t h a t a l l t h e s a l t c r y s t a l s are wetted by the H2S04.

- Leave i t for 10 minutes .

- Add some water and t r a n s f e r i t t o a v o l u m e t r i c f l a s k of 100 ml. ( on t h i s moment the s o lu t i on must be c l e a r . If t he re i s a y e l l ow c o l o u r i t i s n e c e s s a ry t o do a b l ank of t h e sample t o c o r r ec t for t h i s co lour . This sample blank has to contain the same amount of s amp l e and a l l t h e r e a g e n t s e x c e p t s od iumsa l i cy l a t e . The absorbance must be subs t rac ted from Ax.)

- D i l u t e t i l l 70 ml and add c a r e f u l l y 15 ml of 10 M NaOH and mix.

- Leave i t t o cool and d i l u t e ' t i l l 100 ml.

- A f t e r 30 minutes measure t he absorbance (Ax) a t a wave l eng th of 415 nm agains t d i s t i l l e d water.

- Measure a l so the absorbance of the blank (A b l ) .

- Ca lcu la te the co r rec ted absorbance Ax = Ax - A b l .

Calibration.

Pipet from the stock n i t r a t e s o lu t i on 0;1;2;3;4 and 5 ml in a beaker of 50 ml . T r ea t t h i s as a sample and measure t he absorbance aga ins t d i s t i l l e d water. Make a c a l i b r a t i o n curve w i th t h e amount of m i ' s on the h o r i z o n t a l and t he c o r r e c t e d absorbance on the v e r t i c a l axes. The curve has to be l i n e a r .

67

Calculations.

Read from the calibration curve the slope (absorbance/ml), Calculate the calibration factor f = 20/slope

Calculate the concentration according;

[N03-N] = f * Ax / Vo

[N03-N] = nitrate concentration (mg/L N03-N). Vo = volume of sample in mi's. f = calibration factor. Ax = corrected absorbance.

[N03] = 4.429 [N03-N],

7.11 PHOTOMETRIC DETERMINATION OF NITRITE.

By presence of nitrite and acid sulfanil acid will become a diazo compound. This compound together with N-(l naftyl) 1,2-diamino- ethanehydrochloride gives a red coloured azo compound. The absorbance of this solution can be measured at 542 nm. The minimum amount that can be measured is 0.025 mg/L.

Apparatus.

A spectrophotometer suitable for measuring at 542 nm Cells with a path way of 10 mm.

Sample.

Samples for nitrite cannot be stored for a long period. The analysis has to be done within 24 hours after sampling. It is not allowed to acify the sample. Use for the determination 80 ml or less sample containing 2 - 100 ug/N02.

Procedure.

Samples.

- Pi pot the sample (Vo ml) in a measuring flask of 100 rnl (maximum 80 ml).

- Add: 2 ml Na2 EDTA solution (2.2) 5 ml sulfanilamid solution (2.3). 2 ml hydrochloric acid (2.1)

- After 3 min add: 1 ml N-(l- naftyl) 1,2 -diamino-ethane-dihydrochloride solution. (2.4)

- Dilute till the mark and mix.

- Measure after 15 minutes the absorbance of the sample Ax.

68

- The wavelength has to be 542 nm. With distilled water the absorbance reading has to be zero. Measure also a blank made from distilled water and all the reagents. The absorbance is Ab. The corrected absorbance is Ax' = Ax -Ab.

Calibration.

Pipet from the standard nitrite solution 0,1,2,3,4 and 5 ml in measuring flask of 100 ml. Dilute till 80 ml and treat it as a sample. The solutions contain 0;3;6;9;12 and 15 ug N02-N. Make a calibration curve with the amount of ml on the horizontal and the corrected absorbance on the vertical axes. The curve has to be linear.

Calculations.

Read from the calibration curve the slope (absorbance/ml). Calculate the calibration factor

f = 3/slope

Calculate the concentration according;

[N02-N] = f * Ax / Vo

[N02-N] = nitrite concentration (mg/L ) Vo = volume of sample in ml f = calibration factor Ax' = corrected absorbance

[N02] = 3.286 [N02-N]

7.12 BACTERIOLOGICAL ANALYSIS.

7.12.1 Working procedure of the laminar cross flow bench

- Remove the cover on top of the laminar cross flow bench.

- Remove the plastic from the front.

- Switch on the fan and check the LED indication, green light means laminar flow air velocity, is according specifications. Red light means laminar flow air is not according specifications. Check if the filters on top are clean.

- Clean the complete working area with ethyl alcohol.

- Clean also the instruments to be used with ethylalcohol.

- Leave the bench for 1 hour to be sure to have a clean working environment.

69

- Transfer the sterilized equipment tubes and solution directly from the steriliser to the bench.

- Install a burner.

7.12.2 Sampling

Samples must be collected into clean sterilized glass bottles of min. 100 ml capacity, in such a way to guarantee that they are representative of the water being examined. It is important to prevent contamination of the water sample. Sample bottles should not be filled to the birm, since enough space should be left to shake the contents thoroughly before cultures are prepared.

Microbiological testing should take place as soon as possible after the samples have been collected. If the water cannot be tested within one hour they should be well cooled, especially if any lengthy transportation is involved. Samples should be transported in insulated containers and stored in a refrigerator. Even with carefully cooling, no more than 30 hours (Standard Methods) elapse between sample collection and analysis.

There are two ways of establishing the bacterial count in water:

- the MPN (most probable number) method;

- the membrane filtration method.

7.12.3 Decimal Dilution Series

Fill a series of test tubes or conical flasks with 9 ml or 99 ml of water or buffer solution, close with cotton wool plugs or caps and sterilize.

Pipet 1 ml of water sample into a tube or flask and shake to mix. Pipet 1 ml from the first tube or flask into a second tube or flask and again shake to mix.

Prepare the subsequent stages in a similar manner, thereby producing dilutions which differ progressively by one ( 1 + 9 ml) or two (1 + 99 ml) powers of 10.

70

7.12.A Counting methods

MPN METHOD:

Implementation of the MPN (most probable number) method requires that the sample be divided into several (min. 3) parallel dilutions by mixing with a liquid medium. The number of tubes per dilution series exhibiting growth following incubation is matched against table 11 and the MPN, i.e. the most probable bacterial count referred to 100 ml of water sample, is read off. In the case of more than three dilution stages, the highest stage at which all the tubes are still positive as well as the next two highest are used for the evaluation.

If the quantity of sample per tube for the three dilution stages under scrutiny differs by a factor (e.g. 0.1, 10) from the table, the MPN should be multiplied by the reciprocal of the factor (e.g. 10, 0.1).

Table 11. Most probable number per 100 ml for 3 or 5 parallel cultures per dilution stage.

Positive tubes MPN for 100 ml

10 ml 1.0 ml 0.1 ml 3 tubes per dilution stage

5 tubes per dilution stage

0 0 0 0

2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3

0 0 1 2 0 0 1 1 2 0 0 1 1 2 2 3 0 0 0 1 1 1 2 2 2

0 1 0 0 0 1 0 1 0 0 1 0 1 0 1 0 0 1 2 0 1 2 0 1 2

3 3 3 6 A 7 7 11 11 9 1A 15 20 21 28 30 23 39 6A A3 75 120 93 150 210

< 2 2 2 A 2 A A 6 6 5 7 7 9 9

12 8 11

11 1A

1A 17

71

Positive tubes MPN for 100 ml

10 ml 1.0 ml 0.1 ml 3 tubes per 5 tubes per dilution stage dilution stage

3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

3 3 3 3 0 0 1 1 1 2 2 3 3 4 0 0 0 1 1 1 2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 5 5 5

0 1 2 3 0 1 0 1 2 0 1 0 1 0 0 1 2 0 1 2 0 1 2 0 1 2 3 0 1 2 3 4 0 1 2 3 4 5

240 460 1 100

>= 2 4 0 0 13 17 17 21 26 22 26 27 33 34 23 31 43 33 46 63 49 70 94 79 110 140 180 130 170 220 280 350 240 350 540 920 1600

>= 2400

72

MEMBRANE FILTRATION METHOD:

The microorganisms p resent in a defined quan t i ty of water are c o l l e c t e d on an imprev ious membrane f i l t e r , t r a n s f e r e d t o a c u l t u r e medium, i n cuba t ed , and coun ted . F i r s t s t e r i l i z e t h e

-sent) of the _, -p... ,. .,ww^,. ..v. „...,_.,. U41^ u^--ten it by bayonet catch. Pour the water sample into the funnel, openthe tap on the filter base and draw the water trough the membrane filter into the suction flask. Remove the funnel and maintain the suction for 5-10 seconds in order to remove completely and liquid remaining on the filter. Use sterilize tweezers to place the membrane filter on to the surface of the plate (with the underside of the filter in contact with the medium) and incubate. The active substances contained in the i icuiuj iw a n u x u t ; u u a i_t:. m e a u c i v t : b u u b t d i i c e s u u n t t i i i i e u i n u n e nedium d i f f u s e t h rough t h e f i l t e r and e n a b l e t h e b a c t e r i a present on the upper surface to develop i n to v i s i b l e c o lon i e s . ' o r q u a n t i t a t i v e d e t e r m i n a t i o n s , c o l o n i e s a r e counted t o t h e

m< pre F^- -,-«.. -, groups to which they are belonged.

7.12.5 E .coli and coliform detection and count

DETECTION:

Place 100 ml of water sample into a bottle containing 100 ml of double-strength lactose peptone broth and incubate for 4 4 + 4 hours at 37 + 1.5 C. To be sure of detecting Escherichia coli, a parallel culture should be incubated at 42 + 0.5 C. After 20 + 4' hours check for gas formation, colour change (bromocresol purple: from purple to yellow) and turbidity caused by growth. If this check should prove negative, continue incubating for a further 24 hours. If, after this time, there is still no apparent change, this means that no coliform bacteria are detectable in 100 ml water sample. The test is thus concluded.

COUNT:

MPN Determination

Presemptive test: From the water sample, prepare several Parallel dilutions in a decimal series in or, when a large

73

Confirmatory test: From all tubes in the presemptive test where a gas bubble has collected in the Durham tube transfer a small quantity of material with a loop into test tubes of MacConky broth into which a Durham tube has again been inserted. Incubate for 48 + 3 hours at 35 + 0.5'C. For tubes where gas has collected the confirmatory test for the presence of coliform bacteria is positive. Inoculate streakwise on to ENDO C agar or LEVINE EMB agar for identification of the bacteria.

Bacterial counting: The MPN is calculated for coliforms taking the tubes from the 3 highest dilution stages in the exploratory test in which coliform bacteria were detected.

Test for faecal coliforms: Material from tubes which have proven to be positive in the test should be re-inoculated on to EC broth and incubated for 2 4 + 2 hours at 44.5 + 0.2 C. Under these conditions, any gas formation in EC broth indicates the feacal origin of the coliform bacteria.

Membrane Filtration

Filter the water sample, place the filter on to ENDO agar and incubate for 2 0 + 4 hours at 37 + 1.5 C. Coliforms grow as moist, red colonies and are counted according to the visual different groups to which they are belonged. In order to obtain pure cultures, at least one colony from each of the red types is transfered to the respective number of ENDO agar plates to thin as to enable individual colonies to develop.

Filter should be placed following filtration on to a cardboard disc saturated with membrane-filtration ENDO broth and incubated for 22 to 24 hours at 35 + 0.5 C. Improved selective cultivation of the coliforms can be achieved if the filter is first incubated for 2 hours on a cardboard disc with lauryl sulphate broth and then for 20 to 22 hours on a cardboard disc with membrane-filtration ENDO broth. The typical coliform colonies (dark red with metallic sheen) can then be counted.

7.12.6 Detection and count of enterococci

DETECTION

Presemtive Test

Inoculate vessels containing 100 ml of azide dextrose broth with quantities of water sample decreasing each time by a factor of 10. For sample quantities of 10 ml and more use double-strength broth, which is then diluted with the water sample to single strength. Incubate for up to 48 + 3 hours at 35 + 0.5 C.

74

Positive reaction: Turbidity. Indicates enterococci!

Negative reaction: No turbidity. No enterococci present. The test is thus concluded.

2. Confiramatory

From each of the vessels which yielded a positive result in the exploratory test transfer 3 loop fills of material to a tube containing 10 ml bromocresol purple azide broth. Incubate for 24 hours.

Positive reaction: Colour change to yellow, sometimes accompanied by turbidity. Enterococci present!

Negative reaction: No yellow colour. No enterococci present.

COUNT

1. MPN Determination

Prepare at least 3 dilutions of water sample in azide dextrose broth in the decimal series. Work out the MPN based on the positive tubes resulting from subsequent confirmatory test.

2. Membrane Filtration

Filter various quantities of water sample through a membrane filter. Then place the filter on KF streptococci agar and incubate for 48 hours at 35 + 0.5 C. Enterococci grow as dark red to light red colonies. Take the total enterococcal count, with preference being given to those plates with 20-100 colonies. Refer the count to 100 ml water sample.

7.13 BIOLOGICAL OXYGEN DEMAND.

- Prepare a fresh dilution solution.

- Calibrate the oxygen-electrode.

- If the samples have a pH>9 or <5 neutralize with 1 N NANO^or H 2 S0 4 .

- Make 2 dilutions for every sample according to table 12. Prepare about 500 ml.

75

table 12; Dilution table for BOD.

expected BOD in mg/l dilution dilution factor

5 t o 10 1 + 1 2 10 t o 30 1 + 4 5 20 t o 50 1 + 7 8 40 t o 100 1 + 1 4 15 80 t o 200 1 + 2 9 30 160 t o 300 1 + 4 9 50 300 t o 600 1 + 9 9 100

With BOD-values >600 dilute 10 times and use the table again.

- Aerate the diluted sample for 1 minute.

- Immediate fill the BOD bottle.

- Measure the oxygen concentration in the bottle and note the value.

- Close the bottle and be sure that is completely filled.

- Store the bottles during 5 days in the dark at 20 C.

- After 5 days measure the O2 contents again.

- Measure also the O2 content of the dilution-water at start and after 5 days.

Calculation

Dilution water:

BOD = 02 <start> - 02 <5 days> (mg/1).

The BOD of the dilution water must be low and not more than 1 mg/1.

Sample :

BOD ={(02 <start> - 02 <5 days>) -(1 - 1/f) * BOD<dilution>} * f

f = dilution factor

76

7.14 CHEMICAL OXYGEN DEMAND.

- Pipet v ml sample in a COD-flask (see table 13).

table 13; Sample volume for the COD determination,

expected COD sample volume (ml)

COD<750 20

750<COD<1500 10

1500<COD<3000 5

COD>3000 dilute

- Add some glass beads and if necessary dilute to 20 ml.

- Add 2 ml H2S04 18M.

- Add HgS04 (see table 14).

Table 14; Amount of HgS04 to be added for the COD determination.

sample volume mg HgS04

20 30 * CI- contents in meq/1



- Pipet exact 10 ml K2Cr207-solution in the flask.

- Add 30 ml silver containing H2S04. Mix thoroughly.

- Connect the flask to the condensor and reflux during 2 hours.

- Cool and clean the condensor with water. Dilute the contents of the flask to +/- 150 ml and let it cool.

- Add 2 drops of ferroin.

- Titrate with 0.25 N Ferroammoniumsulphate till the colour changes from green to brown.

77

- Note the amount titrated (b ml).

- Do a blank determination with 20 ml distilled water and all the reagents (a ml).

Calculation

COD = (a-b) * t * (8000/V) mg/1 02

V = ml sample. a = ml ferroammoniumsulphate with blank. b = ml ferroammoniumsulphate with sample. t = normality ferroammoniumsulphate.

8. MAINTENANCE OF INSTRUMENTATION,

8.1 GENERAL.

To work on a proper way with laboratory instrumentation and equipment, these need maintenance. This can be a simple cleaning, but it can also be necessary to disassemble an instrument. Special because of the problem of dust maintenance is very important. Without this every instrument will fail after a short period. Maintenance can be done daily to yearly.

Daily.

-Place electrodes in destilled water or in another solution which is recommanded.

-Clean all tubes, beakers, pipets, etc which has been used.

-Check if all gas bottles are closed.

-If there are samples, calibrate the conductivity- and the pH-meter.

Weekly.

-Check level of reference electrodes and if necessary refill with the proper solution.

-Renew the solution in which the electrodes are stored.

-Clean balances.

-Check air filter of the atomic absorption and remove condensed water. If necessary replace the filter.

78

- S u c k some d e s t i l l e d w a t e r t h r o u g h t h e f l o w c e l l of t h e S p e c t r on i c 21 .

Monthly.

-Adjust normalities of all titration solutions and compare with previous ones.

-Clean all tubes used for atomic absorption.

-Check on the methods used in the laboratory.

8.2 MAINTENANCE OF SPECTRONIC 21.

Due to dust on the optical part of the instrument light is absorbed. When no cleaning occurs, the reading will be less stable or it will even be impossible to zero the instrument. To clean the instrument, open the two covers. Inside, at the left of the wavelength setting there is also a small black cover to be removed. Clean the mirrors (5x), lenses (2x) and lamps (2x) as shown in figure 14 with alcohol. NEVER use water or aceton. After cleaning,turn on the instrument and adjust mirror Ma to give minimum absorbance (or maximum transmission). Ma is the mirror with the white label. On this way the maximum of light reaches the detector. Place all the covers (3x) on the instrument again. The detector can be reached from the bottom of the instrument. Clean the sensitive part with alcohol.

FOCUSINO MinnoR

i m_

COLLIMA1ING MIRROR

DEUTERIUM

l»M» « • " • INTERCHANGE MIRROR I

CONDENSER LENS

RELAY LENS

H — I it

w m TUNGSTEN LAMP

^

<- ' '

s

ii

Hi

, - i !

: - ' • >

©

Fig 16 Optical parts of Spectronic 21, which need cleaning with alcohol. M = mirror L = lens

79

8.3 MAINTENANCE OF ELECTRODES.

Silverelectrode; When AgCl has precipitated on the electrode it slowly reacts. Remove this AgCl with a tissue.

Saturated K2S04

referenceelectrode; This electrode must always be filled with saturated I<2S04 up to 1 cm under the felling hole . If tne electrode is not in use, it has to be stored in a saturated K 2 S0 4 solution. If the signal to the mV meter is not stable it is possible that the membrane is clogged with AgCl. This can be removed with a piece of fine abrasive sandpaper.

8.4.ATOMIC ABSORBANCE SPECTROPHOTOMETER.

8.4.1 The burner.

The burnerhead and mixing chamber have to be cleaned every week or after working with saline samples, because salt cristals will deposite on the burner and in the spray chamber, for cleaning the system loosen the retaining ring of the burnerhead, disconnect the two cable and the safety interlock pin. Remove the burnerhead and clean it with water and if necessary with a soft brush. Clean the mixing chamber by flushing with water. Dry the burnerhead and push the head into the mixing chamber. Be sure that the slit in the base of the head engages the positioning pin on the left side of the burner chamberneck . Fasten the retaining ring, connect the cables and insert the safety interlock pin into the safety interlock at the base of the burner compartment.

8.4.2 The nebulizer.

Samples for atomic absorption may not contain solid particles because they can clog the nebulizer however in practice the nebulizer can be clogged by dust or solids not be seen in the sample. In most cases the solid particles are retained at the end of the tube and can be easily removed by disconnecting the tube. If the clogging is in the nebulizer it can be removed by pushing a thin wire in the hole. Only when this does not help, disassamble the nebulizer and clean it with water(see fig.7) If it is connected again it needs re-adjustment.

80

8.4.3 Optical systems.

If the instrument is not in use cover it to prevent dust to enter the instument. If there is dust visible on optical parts blow it away with a dry air bulb. The window surfaces can be cleaned with a tuft of cotton moistened with a dilute solution of mild liquid detergent followed by several rinsing with dionized water, or with ethanol. Never use aceton.

8.5 MAINTENANCE LAMINAR FLOW BENCH.

The frequency of filter replacement will depend upon application, environment and usage, i.e. cabinets running in clean areas will need less attention than those running in a standard laboratory.

First pre-filter

- white fibre matrix, washable once or twice. Regular examine pre-filter for clogging, denoted by a darkening of the white fibres. To remove, simply pull away from frame edges. As necessary, renew filter or wash with ordinary detergent in a flat tray. Rinse and squeeze dry on a flat surface. Shake and replace. Never run a cabinet without a pre - filter as this will affect the life of the HEPA filter.

HEPA-filter. If the bench is used on a correct way the HEPA-filter has a life time of 5 till 10 years. The life time will be less, if the pre-filters are in a bad condition and not regular cleaned or changed. To change the filter, remove the screen inside the bench. Remove the rods before the HEPA-filter followed by the filter. Clean the packing in the bench. Unpack the new filter and install with the black rubber against the bench. Install again the rods and the screen.

81

9. R E F E R E N C E S

NEN 6474 (1981) Water determination of nitrite content. Nederlands Normalisatie Instituut Delft.

NEN 6440 (1981) Water determination of nitrate content. Nederlands Normalisatie Instituut Delft.

Standard Methods (1975). Standard methods for the examination of water and wastewater. 14 th. edition Am. Publ. Health Assn. Washington D.C. p 434-436.

Skoog D.A., Wes t D .M. (198? ) F u n d a m e n t a l s of a n a l y t i c a l c h e m i s t r y . Th i rd e d i t i o n . H o l t , R i n eha r t and Winston. London.

82

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