Determination of the Glucose Concentration of an Orange by Using Spectrophotometer

Determination of the glucose concentration of an orange by using spectrophotometer Introduction Concentration of glucose can be determined by using spectrophotometer. spectrophotometer. Spectrophotometer works by measuring absorbance of a solution at a set wavelength since concentration is proportional to absorbance based on BeerLambert’s law. law. Therefore glucose concentration of an orange can be determined. !"lencross et al. #$%%& "lucose is a si'-carbon monosaccharide found in i n fruits however fruits include inverted sugar that is a combination of glucose and fructose. Therefore glucose was e'tracted by the use of "()-)*) assay. "(+-)*) assay is an en,yme with breaks down sucrose into glucose and fructose. !Coventry niversity #$%&. /n this e'periment glucose o'idase breaks down glucose into gluconate and hydrogen pero'ide !01(1&. 01(1 produced produced in the reaction is broken down by an en,yme !pero'idase& that gives the coloured red dye. The red dye !2uiononimin& was used to determine the glucose concentration of an orange !3edichem&. *t 4$$nm the intensity of red dye is measured and the result will be proportional to the concentration of glucose in the sample. "(+-)*) assay was used to break down sucrose into fructose and glucose. Thus glucose can be measured. glucose +-glucose 5 (1 501o

01(1 5 gluconate

*minophena,one 5 phenol 5 01(1

pero'idase pero'idase

red dye 5 01(

 The spectrophotometer was set at 4$$nm and this was the 6ma' at at which glucose absorbs light best because di7erent molecules absorb light at di7erent wavelength. Thus it’s important to have the right wavelength. /n addition BeerLambert theory states that the absorbance is proportional to the concentration thus the absorbance of a known concentration can be compared to the absorbance of an unknown concentration by reference to the calibration curve. !"lencross et al. #$%%&  The aim is to determine the glucose glucose concentration of an orange by running absorption spectrum of a series of known glucose concentration sample and comparing the absorbance obtained of the unknown glucose sample from an orange.

Method

/n preparation to determine the glucose concentration of sample * B and C the e'periment was carried out as described in the laboratory schedule !Coventry niversity #$%&.

Results Spectrophotometer was set at 4$$nm and the absorbance of known and unknown concentration was recorded !table %& and the spectrophotometer was re-blanked as the absorbance was recorded for each concentration. $ C₆H₁₂O₆ Concentrati on !m3& $ Absorbanc e of A $ Absorbanc e of  !"able #$

#$

$

8$

9$

%$$

*

B

C

$.%#

$.#:4

$.::$

$.%4

$.4#8

$.%%%

$.:98

$.4;4

$.%#

$.##;

$.:8

$.:8

$.4<<

$.%#9

$.4<

$.4#

 The absorption spectrum was plotted to create a calibration curve !%gure #&.  The standard concentration of glucose was used to construct a calibration curve.

$.< $.8 $.4 $. Absorbance

$.: $.# $.% $ $

%$

#$

:$

$

4$

8$

<$

9$

;$

%$$

Concentration of C₆H₁₂O₆ !mM$

!&igure #&Calibration cur'e sho(ing the concentration of series A and series  and un)no(n sample !A*  and C$

 The calibration curve is passing through the origin and the line is linear ! %gure #&. To determine the glucose concentration of * B and C the absorbance of the duplicates !%m3 glucose standard solution& was used. *bsorbance of series * and series B was recorded to obtain a line of best =t and to determine the concentration of * B and C the calibration curve was used. *bsorbance of concentration #$ of series * and B was the same !$.%#m3& !"able #&. *s the concentration of series * and series B increased absorbance also increased for both series. *bsorbance of concentration %$$ !series * and B& was not within the line of best =t compared to the other concentration ! %gure #&. * Concentration !m3& +uplicate % !+%& Concentration !m3& +uplicate # !+#& +i7erence !+%-+#&

B

C

$.##

$.<#

%.$

$.#8

$.98

%.4$

$.$

$.%

$.8

!"able +$  Table # shows the values of unknown concentration sample * B and C. The calibration curve was used to determine the values by reading of the absorbance. *n appropriate concentration was selected which was within the range of the calibration curve and the average was calculated of the two replicates. The suitable concentration was sample * because the duplicates had a di7erence of $.$ whereas the di7erence between the replicates in sample B and sample C was greater. *verage glucose in dilution * > $.%%;4 !$.%#$ %.dp&

Calculation, C?0@1(? A%$$g orange *verage C?0@1(?  !m3&' dilution factor ' %$$!ml& ' %9$ ' %$$ Deight of orange !g&

%$E8

C?0@1(? in orange > $.9< !$.94 %.dp& gA%$$g orange Fatio > %G%G# H "lucoseGIructoseGSucrose  ThereforeG 3ass of inverted sugarG $.94 J # > %.<

Discussion Spectrophotometer was used to determine the concentration of sample * B and C. Spectrophotometer works by the use of visible light to absorb substances at particular wavelength. Therefore the concentration of the substance can be

determined !3eah et al. #$%#&. The dye !2uiononimin& absorbed light at 4$$nm !6ma'& because the dye absorbs light efficiently at 4$$nm !Coventry niversity #$%&. 0aving a suitable wavelength is important because it a7ects how much light is absorbed and the accuracy of the results !"ore 3.". #$$$&. Dhen light is absorbed molecules such as chromophore become e'cited and energy is transferred from the ground state to an e'cited state. The amount of energy transferred is related to how much light is absorbed. 3oreover the amount of light absorbed depends on how concentrated the solution is and the number of molecules present. Thus at high concentration more light is absorbed because molecules can collide more therefore absorbance of glucose is high. !"lencross et al. #$%%&

 The calibration curve is a straight line starting from the origin and gradually increasing linearly. The origin is the value of the blanks. The calibration curve shows as glucose concentration increases absorbance also increases linearly. Beer-Lambert law states concentration is proportional to absorbance hence if concentration increases absorbance also increases. !"lencross et al. #$%%&. /n support the calibration curve is a straight line indicating Beer-Lambert law was met. Concentration of * B C was obtained by plotting wavelength along the '-a'is and absorbance along the y-a'is to create a calibration curve. The calibration curve was used to obtain the concentration of * B and C by reading of the absorbance. The values of the duplicates were not consistent and this indicates the measurements were not accurate. Ior e'ample in sample C the concentration in duplicate % is %.$ whereas the concentration in duplicate # is %.4$ there’s a di7erence of $.8. /t could be because of inaccurate pipetting and =nger prints on the cuvettes.  The =ngerprints on the cuvette can a7ect the amount of light being absorbed because it may interfere with the path of light. 3oreover the angle of light may change causing the molecules in the sample to absorb less light. 3oreover absorbance is the di7erence between absorbed light and transmitted light. The proportion of transmitted light is more likely to be high then the proportion of absorbed light hence absorbance will be small !"ore 3.". #$$$&. *lso some light can be scattered instead of being absorbed. To prevent this the sample should be close to the light detector. !"ore 3.". #$$$&

*bsorbance is the di7erent of absorbed light and transmitted lightG A -

log 10 1 / T 

!"ore 3.". #$$$&

Secondly contamination of e2uipment’s can a7ect the concentration of the sample. Cuvette lids can be used to prevent contamination !"ore 3.". #$$$&. 3oreover weighing out the orange was inaccurate due to rounding.  The aim was to =nd the glucose content in the orange sample. (ranges contain sugars such as sucrose glucose and fructose. The glucose content in the orange sample was $.94g. The average glucose content in an orange is #.<:
Reference Coventry niversity !#$%& %%8B3S Skills in Biomolecular Sciences scheduleG +etermination of the "lucose Content of an (range "lencross 0. *hmed K. and Dang . !#$%%& Biomedical Science )racticeM ('ford niversity )ress "ore 3.". !#$$$& Spectrophotometry and SpectroNuorimetry a practical approachM ('ford niversity )ress

3eah 3.S. and Oebede-Desthead P. !#$%#& Pssential Laboratory Skills for BiosciencesM Diley-Blackwell *leppo. Syria !#$%$& 3edichem 3iddle Past /n vitro diagnostics httpGAAwww.medichem-me.comAoldA3ethodA%-SubstrateA%:Q#$"lucose Q#$)*).pdf  ! (ct :$ th #$%& +ione'Thermo Scienti=c *nalysis of fruit Ruice adulterated with me dium invert sugar from beets %;;# httpGAAwww.medichem-me.comAoldA3ethodA%SubstrateA%:Q#$"lucoseQ#$)*).pdf  !(ct #9 th #$%& Loren Cordian The paleo diet httpGAAthepaleodiet.comAfruits-and-sugarsA!(ct #9th #$%& Steegmans 3. /liaens S. 0oebregs 0 !#$$& Pn,ymatic spectrophotometric determination of glucose fructose sucrose and inulinAoligofructose in foods. Livestrong Katural Sugars in (ranges #$% httpGAAwww.livestrong.comAarticleA#8<$;-natural-sugars-in-orangesA !Kov ;th #$%&