Experiment 1( Introduction)

7/24/2019 Experiment 1( Introduction)

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Experiment 1: An introduction to Biology Laboratory

Objectives:

To expose and familiarize te use of p! meter" micropipette" spectropotometer and #eiging

scale basic tecni$ues and some of teir applications in scientific researc%

&ntroduction:

Te study of cell and 'olecular Biology involves a vast number of metods and tecni$ues as it

studies te structural unit of te living system% Laboratory instrument are devised to acieve

accuracy and precision as it deals #it up to microscopic level% Te micropipettor is one of te

most commonly used instruments in science laboratories% (ipettes are used to accurately measure

and dispense relatively small volumes of li$uid% A small volume micropipettor as a range of

)%*+ 1) mL" mid+range micropipettor can andle 1)+ 1)) mL of li$uid and a large volume

micropipettor can measure up to 1))+ 1))) mL% &t is precision instrument and sould be andled

#it utmost care%

,ile for p! meter is an instrument used to measure acidity or al-alinity of a solution

also -no#n as p!% p! is te unit of measure tat describes te degree of acidity or al-aline

bet#een

scale ) to 1.% Te $uantitative information provided by te p! value expresses te degree of te

activity of an acid or base in terms of ydrogen ion activity% Te p! value of a substance is

directly related to te ratio of te ydrogen ion /!0 and te ydroxyl ion /O!+ concentrations%

&f te !0 concentration is greater tan O!+" te material is acidic 2p! value is less tan 34 #ile

if te O!+ concentration is greater tan !0" te material is basic 2p! value greater tan 34% &f

e$ual amounts of !0 and O!+ ions are present" te material is neutral" #it a p! of 3% Acids and

bases ave free ydrogen and ydroxyl ions" respectively% Te relationsip bet#een ydrogen

ions and ydroxyl ions in a given solution is constant for a given set of conditions" eiter one can

be determined by -no#ing te oter% 5ompare to p! papers #ic cange color #it varying p!

levels% Tese papers ave limitations on teir accuracy and as suc" teir use is limited%

25ompton" 6)164

1



Every cemical compound absorbs" transmits" or reflects ligt 2electromagnetic

radiation4 over a certain range of #avelengt% 7pectropotometry is a measurement of o#

muc a cemical substance absorbs or transmits% 7pectropotometry is #idely used for

$uantitative analysis in various areas cemical engineering" clinical applications and industrial

applications% A spectropotometer is an instrument tat measures te amount of potons 2te

intensity of ligt4 absorbed after it passes troug sample solution% ,it te

spectropotometer" te amount of a -no#n cemical substance 2concentrations4 can also

be determined by measuring te intensity of ligt detected% 2At-ins" 6))84

9igure 1: Basic structure of 7pectropotometers%

Lastly" ove 26)1*4 did mention tat te analytical balance is te common piece of

e$uipment in te scientific laboratory% Tese igly precise instruments can measure do#n to ten

tousandts or even undred tousandts of a gram% Te triple beam balance is a type of scale

tat uses counter#eigts on a tree rider beams to counterbalance te sample in te pan% On te

oter and" modern analytical balances use a complex system of electronic sensors to accurately

mass a substance% ,en #eiging" a #eig boat or #eig paper is used to old te substance

being #eiged and protect te #eiging pan% Before #eiging a substance" analytical balances

are tarred to subtract te #eig boat or #eig paper and re+zero te scale% Tarring refers to

setting te scale bac- to read zero and allo#s for a substance to be accurately #eiged% Aside

from #eiging cemical substances" analytical balances are used to animals or insects" and

centrifuge tubes in ultracentrifugation experiments%

2



'etodology

(art 6:

(art A: ;isual Estimation of p!

)%1 ' solutions 21))mls4 of te K 2HPO4 and KH2PO4 was prepared and set up accordingly

followed the Table A-1.2A

drops of !ro"othy"ol !lue was "i# in the tube of 1$ %$ $ &$ '$ and 11. The changes of color was obser(ed and recorded.

7ection A:

%



<sed p! 3 buffer to standardize te p! meter and rinsed te electrodesalso measured te p! using standard p! buffer .%)1%

Te p! of te six solution of 6" ." 8" =" 1)" and 16 tubes 2as prepared in

(art A4 #as measured% Te electrodes #as -eep rinsing in bet#een tesolution%

9or te same solution" p! indicator paper #as used to measure te p! asrapid metod for te small volumes or radioactive solution%

Te measured value from part A and B #as observed and recorded andcorrelate te value from part B to te expected p! value from te table A+

1%6A%

7pectopotometer is#armed up about 6)minutes and set up at

te range *.) nm%

1) mls of distillled#ater #as pipette in

eigt tubes

Bromopenol blue21%6*> #?v4: O%*" 1"6" ." 1)" 6)" *)" and1)) @l #as added in

te tubes by using temicropipettors%

Tespectropotometer#as set to zero #it

distilled #ater and tedye solution ad beentransferred from least

concentrated to mostconcentrate into te

same cuvette%

Te reading valueobtained from tespectropotometer

#as recorded

(art :

(art .:

4



Basic #eiging function: Turned on te balanced scale%

5ontainer #as placed on top of te balance" balanced #eigts to zero ten temsample #as placed in te container%

,eigt of te sample #as recorded%

esult

(art 6:

9igure 6: p! indicators results using Bromotymol Blue%



9igure 6%1: p! indicator color range%

Table 1: 5omparison of p! results using t#o different metod%

7olution 2tube4 p! meter p! paper

6 *%.* *

. 8%6. 8

8 8%8* 3

= 3%)= 3

1) 3%* =

16 =%61 C

(art :

Table 6: ,avelengt results for different concentration solution%

7olution Bromopenol blue

concentration2@l4

Absorbance value

2nm4

1 )%* )%))8

6 1 )%)1)

6 )%)=6

. . )%)16

)



* 1) )%)6=

8 6) )%)8.

3 *) )%1*

= 1)) )%6=3

*+ , -.'

Absorbance (abs) versus concentration of bromophenol blue (w/v)

Absorbance

inear /Absorbance0

9igure : Absorbance 2*.) nm4 versus concentration of Bromopenol blue 2#?v4

(art .:

Table : ,eigt of micropipette results%

Amount micropipette 2@l4 ,eigt 2g46)) )%6)16

*)) )%*)1)

3)) )%8C.3

1))) 1%))=1

&



Discussion

p! is te unit to describe te degree of acidity or al-aline 2) to 1.4% Te objective of tis

experiment #as to expose on basic s-ills te use of p! meter and also p! indicator% Based from

te result obtained in figure 1 so#s different range of p! color from tube 1" " *" 3" C" and 11 by

mixing eac tube #it te Bromotymol blue% As obtained" te comparison bet#een figure 6

and figure 6%1 so#s tat te tubes 1 and solution #as yello# color means te value of p! is *"

#ile for te tubes * and 3 solutions so#s te p! value #as 3 and for te tube C and 11 turned

to blue color indicates te p! value #as C% Bromotymol blue solution commonly used as p!

indicator% Bromotymol blue canges color over a p! range from 8%) 2yello#4 to 3%8 2blue4%

Tube C and 11 turned in blue color because te levels of acid in solution #it Bromotymol blue

indicator #as lo#" for te tube * and 3 its turned to te mid p! range bet#een acidic and base

#ile te level of acid increases" te solution #ill gradually ta-e on a yello# as so#n in figure

6%

Based from te results obtain in table 1 so#s t#o different metod use to indicate te p! by

using te p! meter and also p! indicator paper% 9rom table 1" te value recorded from tube

solution 6" ." 8" =" 1)" and 16 so#s sligtly different from te table A+1%6A% Te value #as near

to te expected p! value for eac tubes solution for an example tubes 6" te p! recorded #as

*%.* near to te expected value *%*C #ile for te solution ." te p! meter value #as e$ually

same to te expected value% Tis is because te p! value of a substance is directly related to te

ratio of te ydrogen ion /!0 and te ydroxyl ion /O!+ concentrations% &f te !0

concentration is greater tan O!+" te material is acidic 2p! value is less tan 34 #ile if te O!+

concentration is greater tan !0" te material is basic 2p! value greater tan 34% &f e$ual amounts

of !0 and O!+ ions are present" te material is neutral" #it a p! of 3% !o#ever" based from te



p! paper indicates tat te value obtained #as more different to te expected value at table A+

1%6A% Tis is because te p! paper or Litmus paper is often used to differentiate an acid from a

base% ,en p! is less tan .%*" te paper turns red% Tis indicates tat te substance is an acid% &f

p! is greater tan =%" te paper turns blue" indicating tat te substance is a base% &n neutral

conditions" p! paper is a purple color% Based from tese metods of p! measurement" te

more accurate metod to measure p! is by using p! meter because te value of p! meter

because te value obtained #as almost same and nearly to p! expected%

9rom experiment part tac-les on te familiarization of t#o commonly used laboratory

e$uipment spectropotometer% A specific amount of Bromopenol blue #as dropped and mixed

#it eac of te eigt test tubes% Based from te table 6 by using te spectropotometer" te

absorbance for )%*L of Bromopenol blue #as )%))8nm" for 1%)L Brompenol blue it #as

)%)1)nm" )%)=6nm #as recorded for 6%)L of Bromopenol blue" for .%)L Bromopenol blue

#as )%)16nm" for 1)%) L Bromopenol blue so# )%)6=nm" for te 6)%) L te results so#

)%)8.nm" #ile for te *) L so# te result obtained )%1*nm and lastly for te 1)) L te

result #as )%6=3nm% Te results so# tat tere is a direct relationsip bet#een te amount of

dye in te solution and te amount of ligt absorbed by te solution% As te amount of dye

increases" te amount of ligt absorbed also increases% 9or te 1)) L" te solution #as more

concentrated tan oters it #ill ave a very ig absorbance because tere are lots of molecules

to interact #it te ligt rater tan )%* L te value #as expected as te concentration #as lo#"

so tat te interaction bet#een te ligt and molecular #ill be less% ,en potons encounter te

molecules in te sample of te solution" te molecules may absorb some of tem" reducing te

number of potons in te beam of ligt and decreasing te intensity of te detected signal% Tis

means te iger te concentration of te solution te iger te absorbance of te #avelengt

#ill be%

9rom te absorbance" te concentration of te sample solution can be determined #ic states

tat tere is a linear relationsip bet#een te absorbance and concentration of a sample%

Based on figure so#s te plotted grap of te amount of te absorbance versus Bromopenol

blue so#s te linearity of te absorbance #ic so#s te iger te concentration of te

iger te absorbance% !o#ever" tere #as an error occur at solution #ic te absorbance

'



obtained #as )%)=6" tis error migt cause from #rongly micropipette te amount of

Bromopenol blue and maybe because of te cuvette%

Lastly for te part . experiment so#s tat te value recorded in table so#s four different

amount of solution by using micropipette% Te purpose of tis experiment #as to familiarizestudents #it te operation of te analytical balance% Because all analytical direclt based on te

measurement of mass% Te volume of distilled #ater #as measure by using micropipette #it

volume 6))L" *))L" 3))L and 1)))L% Based on table " #en te volume of distilled #ater

increase" te #eigt of distilled #ater also #ill be increase% Tis is due to te density of #ater

increase%

1



5onclusion

Te objective of tis experiment is to expose students #it p! meter" micropipette"

spectropotometer and #eiging scale basic tecni$ue% Tis experiment #as acieved te

objective% 9or p! measurement experiment te uses of steps to measure p! by using drops of

Bromotymol blue into te buffer solution" p! meter and lastly p! paper it can be concluded

tat p! meter is more accurate to measure p! of te solution compared to te t#o oters

metod% &n spectropotometer experiment te result so#n in figure" te absorbance of solution

increase #en te concentration of Bromopenol blue solution increases% Te results so# tat

tere is a direct relationsip bet#een te amount of dye in te solution and te amount of ligt

absorbed by te solution% As te amount of dye increases" te amount of ligt absorbed also

increases% 9or te 1)) L" te solution #as more concentrated tan oters it #ill ave a very

ig absorbance because tere are lots of molecules to interact #it te ligt rater tan )%* L

te value #as expected as te concentration #as lo#" so tat te interaction bet#een te ligt and

molecular #ill be less% Lastly" for part #eiging balanced" #en te volume of distilled #ater

increase" te #eigt of distilled #ater also increase tis is because te density of distilled #ater

also increase% As recommendation proper measuring and using te micropipette #as important to

get te accurate result and te cuvette use for te spectropotometer must be in proper condition"

#ipe te fingerprints at te clear parts%

11



Fuestion

(art 1:

14 +Do not allo# te tip of te micropipette to touc te receiving li$uid% 7ome of te li$uid

may be dra#n bac- up into te micropipette" contaminating te sample for te remainder

of te micropipette se$uence%

+Gever old te micropipette side#ays #en li$uid is inside te tip%

+Gever flame te tip of te micropipettor%

64 5are :

+Disassemble and clean te pipette

+emove te tip ejector 7eparate te lo#er alf of te pipette from te top alf

+Decontaminate eac component as it is removed from te pipette

+emove te seal and o+ring assemblies

+&nspect visually for #orn and crac-ed parts

'aintainance:

+Treat micropipettes very gently as tey are precision instruments%

+Heep uprigt #en in use to prevent li$uids running inside te saft of te pipette%

+Do not leave pipettes lying on te #or-benc #ere tey can be -noc-ed off and

damaged%

+5ec- all tips are securely fitted to pipette%

12



(art 6:

14 A p! meter provides a value as to o# acidic or al-aline a li$uid is% Te basic principle

of te p! meter is to measure te concentration of ydrogen ions% Acids dissolve in #ater

forming positively carged ydrogen ions 2!04% Te greater tis concentration of

ydrogen ions" te stronger te acid is% 7imilarly al-ali or bases dissolve in #ater forming

negatively carged ydrogen ions 2O!+4% Te stronger a base is te iger te

concentration of negatively carged ydrogen ions tere are% A p! value of 3 indicates a

neutral solution% (ure #ater sould ave a p! value of 3% Go# p! values less tan 3

indicate an acidic solution #ile a p! value greater tan 3 #ill indicate an al-aline

solution% A solution #it p! value of 1 is igly acidic and a solution of p! value of 1. is

igly al-aline% A p! meter #ill be made up of a probe" #ic itself is made up of t#o

electrodes% Tis probe passes electrical signals to a meter #ic displays te reading in

p! units% Te glass probe as t#o electrodes because one is a glass sensor electrode and

te oter is a reference electrode%64 5alculation:

GaO! 6)> #?v

;olume 6)) ml20

100×200ml=40

g

mol

Tris 1%) ' 2', 161%1 g?mol4;olume 1))) ml

1000ml× 1.0mol

1000ml×121.1

g

mol=121.1 g

EDTA )%6 ' 2', 36%6 g?mol4

;olume 1)) ml

1%



100ml× 0.2mol

1000ml×372.2

g

mol ¿7.44 g

4 5alculation:

Tris 1) m' 2', 161%1 g?mol4;olume 1)) ml

100ml× 10mM

1000ml×121.1

g

mol=0.12g

EDTA 1 m' 2', 36%6 g?mol4

;olume 1)) ml

100 ml×

1mM

1000ml ×372.2

g

mol=0.037g

.4 Te buffer capacity depends on te concentration of acid or base in a buffer% 9or a good

buffer" te acid concentration to be about te same as te base concentration% Tus" te

closer te acid and base concentrations are to eac oter te more tey approac 1 #ic

results in a log 214 #ic e$uals )% 7o good buffers mean teir p! #ill be around pHa%

Tis also means a better buffer capacity%

*4 Buffer capacity defined as maximum amount of eiter strong acid or strong base tat can

be added before a significant cange in te p! #ill occur depend on o# ig teconcentration of te acids and bases are in te buffer% Tis is because if te

concentrations of acid?base are extremely large" tan it #ill ta-e muc more of an added

acid?base to cange muc of te bufferIs acid?base% Buffer capacity can be also defined as

$uantity of strong acid or base tat must be added to cange te p! of one liter of

solution by one p! unit%

(art :

14



14 A spectropotometer consists of t#o instruments" namely a spectrometer for producing

ligt of any selected color 2#avelengt4" and a potometer for measuring te intensity of

ligt% Te instruments are arrange so tat li$uid in a cuvette can be placed bet#een te

spectrometer beam and te potometer% Te amount of ligt passing troug te tube is

measured by te potometer% Te potometer delivers a voltage signal to a display

device" normally a galvanometer% Te signal canges as te amount of ligt absorbed by

te li$uid canges% &f development of color is lin-ed to te concentration of a substance

in solution ten tat concentration can be measured by determining te extent of

absorption of ligt at te appropriate #avelengt%

64 7pectropotometers are instruments tat are commonly used for measuring te

transmittance or reflectance of solutions or transparent% Tis instrument is basically used

in field biocemistry because <; visible spectropotometers are used in te $uantitative

determination of solutions of transition metal ions and igly conjugated organic

compounds%

4 Tere is a logaritmic dependence bet#een te transmission of ligt troug a substance

and te concentration of te substance" and also bet#een te transmission and te lengt

of material tat te ligt travels troug% Te percent transmittance multiplied by 1))" is

usually converted absorbance%

eferences:

1



5ampbell" '%H%" 9arell" 7%O" Biocemistry% 8t ed% (ilippines: 5engage Learning Asia(te% Ltd%

26))C4

At-ins" (eter and ulio D%(% (ysical 5emistry for te Life 7ciences% Ge# Jor-: Oxford

<niversity (ress" 6))8

5ang" aymond% (ysical 5emistry for te Biosciences% <7A: <niversity 7cience Boo-s"

6))*%

Kore" 'icael% 7pectropotometry 7pectrofluorimetry% Ge# Jor-: Oxford <niversity (ress"

6)))%

1)

Experiment 1( Introduction)

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