UPH-OR. JOSE G.

TAMAYO MEDICAL UNIVERSITY

COLLEGE OF MEDICAL TEC HNOLOGY
CLINICAL C HEMISTRY 1

ACTIVITY 3
SPECTROPHOTOMETRY
LEARNING OBJECTIVES
,,., IM coodualon ol \hlS act,vily. lhe lludenl should be able IO
1 explain the principle of speC1roph01ome1ry
2 descnbe the ope!abOn and component pans of a •pectrophotometc,
3 care and precaution a whe n using a 1pectropholomete1.
• · know the principle of Beef s Law (also known as Beer-Lamber1's Law)

PRE-I.AB DISCUSSION
Many determinations made tn the clinical laboratory are based on the meaaurement of
,adtant ene,rgy ab5ort>ed O< transmitted under controlled cond~10ns. The device used to meas-
ure the ltbM>rbed or lranamilted llghl e n ergy ts the spectropholomell!r
Spectrophotomete< Is used 10 measure lhe tight tran,wmltt!<I by • 50iut1011 In O<det to
c1e111rmme the concentration ol the ligh t-ab1elbing substance In Lhe .olutlon. Thos determ,na-
llon ,s mlldle by passing a narrow beam of llghl lhrough lhe solution light i1 a form ol elect,o-
magr,euc radiation (EMR) conlil1llng of energy travet,ng ,n waves. EMR consists of gamma
rays. 1-raya, ukraviolel (UV), vlalble. Infrared (IR). microwaves. and rad,o waves
The ponlon of light that pa sses through the colored solution 11 the percenl tran5ffltt-
tanc:e (%T) The hght thal does not pan thrDY9h Is absorbed by lhe solution and 11 moeaure<!
as a~rbal\Ce (A) un~s
These aolul10ns 11111 said lo follow Beefs Law. a mathematical relationship thal 11818.5
t11a1 me cana,ntratlon of a substance Is rluectly proponlonal to lhe amount of light ablOrbc!d ,or
,nv&rsely p roponlonal to \he logarithm of the transmitted llghl

EQUIPMENT and MATERIALS

Spectropholometl!r Model 722
Square Cuvetles
SISndan:l and unknown sample solutions

OPERATION OF THE SPECTROPHOTOMETER

1 Allow 11'\e spectropttDIOmeler lo warm-up for 15 minutes bef01e uaing

2 Adjust the desire<1~_:w:a:ve1e:::&:~:=~'h:.;;".,;,;w,.'""11,iiiiileiiinoiLln...,1'n11o111b.,.-""II

•

e
l2
 UPH.OR. JOSE G. TAMAYO MEOICAL UNIVERSITY
COLLEGE OF MEOICAL TECHNOLOGY
CLINICAL CHEMISTRY I /

open me sample cover

3

• --1 ISample Cover I

-.-
I
•I
-----1!--I Sample Compar1ment

j DrawRod I
, Prepare three square cuvenes

5 Dispense the blank ,nto lhe first cuvelte. the reference staridard Into Ina 5eC00d
cuvette, and the unknown sample Into lhe third cuvette

33
 CLINICAL CHEMISTRY I

ACTIVITY 3
SPEC1lt0flt40TOMETRY

Nmne·-
ui,onatory- - -- -__
lnstrudors· -_
-_-_
-_-_
-_-_
-_-_
-_-_
- _-_-_
-_
-_- -~ ·:_:_:;=:_:_:_:__----
o_a_,e_s_u_b_m_11_,ea
__

QUESTIONS
1 E,cplaln the pnnciple of 1pe<11ropholometry
--
2 Give the 111nge of wavelengths (nm ) p Uhravlolet , Vi1lble and lnfrarea &peclNm.

3 Slate the pnnciple ol ttie Beer-Lambert Law and give the mathemaliceJ formula.

J Draw a diagram of the Internal pan_s (components) of a basic spectrophOlomeler and

labet each pan

5 Give the pnnaples of the following instrumentalions and their dlnlcal applicatJons
Use the back of this page for your answe1s
A. Atomic Absorption SpectrophOtometer
B . AUOIOfflBter
C . !Flame Photometer
D. Nephelometer
E T urt>idimeter
F. Flow c)'IOme1er
G. Eledn,phoefes!I
H Chromatography
1 Mass Spectrometer
J . Sdnlillalion Counters

s . Give the different type• of cuvenes used In spectrophotometry and the"

corresponding usage

7 Why do you use 8 blank solution? A standard?

__.,
8. G,ven a ,.,....,ng of SS% Ton the spectrophotometer. convert 10 Ab1orbanee

...,.....,pholometers Name at loaSI r,,~ calibliltJOO

9 Why do you
have t o calibrate the s,...--
_,.. gth of each at the point ot callbratoon
.
materials and the wav=n

35
 UPH-OR. JOSE G. TAMAYO MEDICAL UNl~ ERSITY
COLLEGE OF MEDICAL TECHNOLOGY
CLINIC AL CH EMISTRY I
ACTIVITY <I
GLUCOSE DETER MINATION
0 -TOLUIDINE METHOD (DUBOWSKII

f,\llNING OBJECTIVES
~ u,<t a,,,cjuSIO<' ol INS Ktlvrty' lhO SIUdl!fll ShO<Jlcl l>e Obie lo
1 pe,tom, Ilic! 0-Tolulcllno Method for olucoMO delorm,na1,c,n
: i.r,ow \he pnnciplel ot 01he1 c;hem,c.al method• for glucose teals.
J 11, - t"e pru,c:,ples ol on..-ymnuc mothO<I• tor glUCO'J0 delerm,na\Jons
i . . , _ 1ne cllnlcal s,grofic:ancc ot 01uco&11 dnlermlnolion •

,,;tE.US OISCUSSION
GIUCO$<! rs meuured In whole blood plasma, serum CSF p!iltural fluid and unne
c;tucoff me1hoJs can bo dMded Into \WO g roul)$-Chemteal and onzymabc Or1'>~
l(!lu,IS,'"' is IN only chemlCAI method still used today and•• based on the con<lenutlon at
~ andet t ueh u glucose W11h an aromatic amine and glacial «-le add The liable
.,,_, c,o1o< u,m develops u,c,n Is me»ured spectrophotomelrically
Er1--rna1c m ethods yield m;ulmum spec1f1cily f or glucose· mea■urements Three
,-:,me J)")IIIMS 11re currently used to m easure glucose glucose d ehydrogenase. glucose o,o.
:JSlf - ~e
~E
O-toiucl<ne - n heated al 100° c ,n glacial acetic aoct forms a green derivative WII"
~ ..,.,, a,, abso<t>anoe ma•lmum of approJCJmalely ~O nm Ttle reaetion i5 reasonably

SAMf'l.E: P1asma or unhemolyzed serum

REAGENTS AND MATERIALS

Gluco-lolultllne Reagen\ Centrifuge
Glucose Standard - t 00 and 200 mgldl Water Bath
Si,earophot.o meter

PffOCEDURE
Prepare 1wO test 1ube11 and labe4 eKh as Standard and test: or oonlro(lf avalable)
Sblndard 100 Of' 200 mg/dL Te•t Senl Of' Contrala
REAGENTS
Gtucaae Stand•rd 0.1 ml -
Tnt SeQ/Control ------·-- 0.1 ml
3.0 ml
Gkle~lne Rugent 3,0ml

2 Miwwell and pi.ce a1 bo4IIng wato, at the same ume (Standatd and TnVcontrot)
fo r 3 (lhteel mInU1e• (Make 11uA1 lhlll \he water bath ,s v;gorously t,olllnQ)
3 Cool
Read 1110 $ pec111)PhOU)rTleler Wavelength IS 61 ~ o nm
4 111
S Read
· ..,, p · • wlll••
_...,nil blanlo.
r--111ance(%T) antl Ab!IOf1>ancei A l ol lhe •
..,,i ,
.,
W 'l>d
6 Re ad the e rcen1 •-•-"

CALCULA TIOH OF RESULTS

A of the Unknown/Tes, )( Value ol the x 0.05S
Stan<1erd
Value of the unknown/leSI • A of lhe Standard
(mmoVll

REFERENCE INTERVAL tH■nry

21 s1unlt
~ 0~~-! nllonal Unit 39-61 mmoVL
Spechnen 70 - 1 i OmQldL 2.a - 4 4 mmoill
Pla&ma1Serurn(Fas1Jng) 4 o _ BO mgldl
S pmal Fluid

36
 - - - . ... - ~ . ... ...... I 1:1...HNULOGY

CLINICAL CHEMISTRY t

ACTIVITY 4
GLUCOSE DETERMINATION

~
L,al)OlalOIY 111111NC1or1
QU£ST10NS
----------------========----
- - ~- - - = = - - - - -- - - - _ Date Subm11ted

G,ve at least 4 metl'Klds whoM principle falls under Alkaline Copper Redlldion Test
Whldl of tills tesl la spaoft<: for glucose?

2 c;.-. ih11 pnndple, 1nvoived ,n lhe o>dda11e and he,okmue enzymatJc melhods
tor glucose lltultrate the chemoc.al reacllon or equation of bolh methods.
\\ltuch is the generally acceptance method lo, glucose detenninallon?

3 Enumerate \he subsUlnces wtuch can cause falsely dec:teased values In gluC0$e
oxkiase assay

• Explain the procedure ol 2-hour Post-Prandlat Tell and Oral Glucose Tolerance
Test Give the reference values.

5 Whal 11 1110 usoful 1echnlquc lor aucSSlng the loi,g term control of diabtrtcs mellttu,?
Enumerate me mc:nods. 'Mlot Is lhe Referena, Value?

·mena should be separated Immediately from th~

6 Eltplain why 1ne glucose.hspeof rred vacuum cotlec:tlon lube 1ha1 can be used to
red b,oodlce
pravent t
i "'nd~:'
1• a
lspr~a:!':.,:e lhBI 1nhlbl15 11115?

of metabolism of glucose at room tempe,-ture?

7 11\/lUll IS lhO COle

37
 UPH-DR. JOSE G. TAMAYO MEDICAL UNIVE
COLLEGE OF MEDICAL TECHNOLOGYRSITY /
CLINICAL CHEMISTRY 1

ACTIVITY 5
TOTAL CHOLESTEROL DETERMINATION
UEBERMANN•BURCHARDT MODIFtEO
LfA/INIMG OBJECTIVES
Al IM a,ndusion of this actlvhy' the studenl ahould be •Ille lo.
1 perform the Lerbermann-Burchardt ~hod for cholesterol -rmlnllbOn
2 know the principles ol other chemical methods for chole■terol tests, ·
3 know Ille p:tnciplH of enzymatic metllods for cno1e■tero1 determinations.
• i.now ine dlnk:al slQnlflcance of Cholesterol determination

PRE~ DISCUSSIOtf
ColO(rmetnc procedures lor the chemic.al quanlltalJOII ol serum cholesterol ls the 0 ...-
slep o,red metnod ol Ille Ucbermann-8urchardt procedure Cholesterol (ln both lree and ester
ro,rns) ~ with a mixture of sulfuric acid and ac;etic anhydride 10 form oxldetlon p,oduas
(green end-COior) wlllch absotb strongly in the regron ol 610-640. The strong acid combinalion
;en,o,;es the hydroxyl group and introduces another double bond into lhe 51er0id nng. thereby
,nereasing the color intensity

SAMPLE: Unhemolyzed MNm

REAGENTS AND MATERIALS

ChOlestefOI Reagent A Cemrif1111e
C ~ Reagent 8 Spec11ophot-
Cholesterol Slandard - 200 mgldl

PREPARATION OF CHOLESTEROL WORKING REAGENT

1 UH oven dry g!UI ~t.!nor 1114 gl'ldy!lted cylinder.
2. Add one pert ol Reagent B lo one part of Reaoe,,t A .
3. Mix well by swllfing and cool lo room temperature before ualng
4. This stable IIOlution is ltable for six months in • refngerator.
NOTE: Avoid using wet pipettes. test tubes and cuvettes when pecformrng the test

PROCEDURE
1. Prepare two test tubes and label each as Standard and test o, conbul(il ev■'1ablel

REAGENTS STANDARD 200 mgs. TEST SERA Cl# CONTROLS

Cltoles1arol Standard OJ15mL

Test Sara/Control - O.Hml

3.0 mL 3.0ml
Cholestlrol Working Reagent

2 Mix -a by swlrtlng or use a Vortex ml<er

3. Stand Ill room tempent1ure lor 3 (three) m,n~\es6 I 0-640 nm
• · Read in the Spectrop1101ometer Wavetengl s
5 Read aoainSI a water blank ol ~ lellSI
6 Read the Percent Transmrnance('l<oTl and AbsOrt>ance{AI u,e

CALC~ OF RESULTS
st
A ol the UnllnowntT• X v alue of the x o 026
Value ol the unknown/lest" the Standard S111ndatd
(mmoVLl A 01

2 SI unit
REFERENCE 1NT£RVAL (Hanry ~!~~nal Unit 3 ea _ 6 4 7 mntolll
Total Ch~l8f'l)I t 50 - 2SO mgldl
Serum(Fasbnll)

38
 UPH-DR. JOSE G. TAMAYO !MEDICAL UNIVERSITY
COUEGE OF MEDICAL TECHNOLOGY •
CLINICAL CHEMISTRY I .•I
I

ACTIVITY 6
HDL- CHOLESTEROL DETERMINATION
IIAGNESIUM-Ptt<>SPHOTUNOSTATE/UBERIIANN BURCHARDT MODIFIED
~NING OBJECTIVES
~1ine c:ondu110n or 1h11 activny. lhe s1ue1en1 should be able 10
1 peitom1 U,e magnesium-phospahale method for HOL-Cho&es1ero1 ClelflmlonlllJOn
2. know lhe pnnciples Involved In lhe various mthods for HDL·Chole11ero1 auay. ·
J knOw lhe impor!Ance ol HDL In lipid lranaport In 1he body,
• · know lhe donital ll9nilicance of HOL delermlnallon

PllE·LAB DISCUSSION
The measuremen1 ol h,gh densKy llpoprolel n (HOL) involv1ls 1WO basic 11ep1.Flrs1, 01her
llpOPfOlrein c18S!ff mull be teparllled from the HOL proteins Seaind. choleslerol mu11 be
qulW11~
The separa1Jon process can be accompliahed With the uae ol varioua preclpRaUng
q,,Is 10 remove VLOL and LOL componen11 of lhe mildure Several combinations ol polyan.
IQIMllvlllen1 such as hepann IUllate-Mn' " , dextran sulfate-Mg'' and sodium phosplKJlu,,gsta.
r.ig:• These precipltan11 remove 1he unw■med proteins. followed by qu■ntution of the cho-
tellelOl ln lhe HOL IIKllon usi119 colotrimetric assay
j,AMPLE: Clear and unhemalyzed serum
-, ~EAGENTS ANO IIATERIALS
Cholesterol Ruger,1 A and B Spectrol)holometer
HOL Predpi1a!l119 Reagent Cen1nfuge
Choles1erol S1andard 50 mg/ell
PROCEDURE A
i . Preparation of Cholfft■rol Woflllng Reagent
1. Use oven dry glass container (beaker) ano gradualed cyflnde1
2 Add one part of Reagent B 10 one par, ol Reagent A.
3. Mix well by swirling and coot lo room temperarure before u11ng
• . Thia ■table IOlutlon Is stable lor .ix mon1hs In a refrigerator
NOTE: Avoid u11ng wel pipettes. test lubes and cu-,ette1 when perlomllng lhe Im
II. VLOL and LDL Precipitation
1 Add 1.OniL ol 1he precipllat,ng reagent to 0.5 mL ol lhe lest aera/controls
In 5 ml teS1 lube
2 Shake ~igorotJsly and centrifuge for 5 (love) minutes at hogh 1peed.
3 Save lhe 1upernanan1 solution (HOL)
PROCEDURE 8
1 Prepare 1w0 test tubeS and label each as Stanaard and test or control(il available)
Reaganu Standard 50 mg/ell THI Sen/Controla
Cholesterol 81andlrd 0.1 ml --
T111 Sera/Control Supem■t■n1 ---- 0 .1 ml
Cholesterol Wo11d119 Ra■gent 3.0ml 3.0ml

2 Mix -11 by ■wining or UM • Vonex mo,er

3 Stand 111 10Dm tempe<11ture for 3{three) minute• •
4 Read !ti the Spectrophotometer Wavt1l1tr>gth ,, 610-&40 nm
5 Read against ■ w111Te, lll■n~cr(%T) and AbsOIDlll'ICll(A) of tl1e !lllrdanl. w ,tra
6 Read 1he Percent ra1'l11Tl

C~CULATION OF RESULTS ._ of t•e Un~nown/Tesl

" X VIIUe ol Ille X 0 026
Value of the unonown/tesl = - -- - - - - - S,.ndard
(mmoll\.l A of Ille Standard

''
40
 UPH·DR JOSE G. TAMAYO MEDIC
COLLEGE OF MEDICAL TEC~OUL~~;RSITY /
CLINICAL CHEMISTRY I
ACTIVITY 7
TRIGLYCERIDES DETERMINATION
BUCOLO •ncl DAVID IMPROVED
LfARNING OBJECTIVES
41 lhO C<)<1dl,11011 of lhll lldlvlty. lhe Sludelit lhOuld be able 10
1 p@11orm lhe_Bucolo end David lmpn:,,11<1 Method fO< lr,glyceride determinallQfl
2 ~now lhe pnnc,plea ol other chemical method• fer 1nglyCllnde IHU ·
3 know 1he princlplea of enzymatic methods lor ltiglyceride detenn,n•s
~ ~now lhe clinical significance of lnglycetid& datermlnati.,,, ·

PIIE~B DISCUSSION
Measuremenl of lriglyceridn (w hether by chemical or enzvmatlc methOda) Involves u,.,
~ 1o1iow,ng general p'OCeS1e11
1 hydrolysls cl tnglycerlde 10 lorm glycerol plus !tee fatty acids.
2. measurement er glycerol 1><esent (either as glycerol 0< after conversion 1o
an01t1er product
The use of enzynw:s for lhe quantttahon of tnglycerides allows a 01111-1191> procedure
""''n ebm10ales the eidractlon slep. The hydr01ysis or lriglycerlde• wtth strong b... is not n=
essa,y since this sl!!p can be accomplished enzyn,at,cally, Glycerol formed from lhe oreak-
oown of 1rlglyoerides lhen 1Nct1 directly wtth an 1nd1ca101 system In • series of enzyme 11!8C·
uons The actual product measured Is either NAO " or NADH Lipase•• empjoyed u lhe en-
zyme rcr hydroly.ls ol triglycerides. producing glycerol plus free fatly acids

SAMPLE: Clear and unhemolyzeo serum

REAGENTS AND MATERIALS
Tnglycende Reagen! Tnglycende Slandard 100 0< 200 mgidl
Triglyceride Buller Reagen! Speciropholcmeter '
Tr,glyoeride Color Reagen! Centrifuge

PROCEDURE
1 Prepare two tesl l ubes and label each as standard and last or control(d •vllilable)

REAGENTS STANDARD TEST SERA or

100 mg/dL or 200 mg/dL COHTROLS
Trlglyuride Standard 0.05mL
Tnl Sera/Con!IOI -···· - - 0.05ml
Ttlglycerida Reagent 1.0ml 1.0ml
Colo, 1tNQ41nt 1 drop 1 drop
2.0ml 2.0 ml
Bu"9, Reagent
2 M It by rtlng or use a Vorlex muu~r
IX we SWI 1 5 (live) minut. .
3 Stand at room 1empenoturo ; WaveJengm ,5 460 nm
4 Read In the SpectrophOlome '
5 Rea d against a w81er btanl ~ (% T) and A~rbance(A ) ol lhe Sland,Jrd. ll!i-,scc,x,nvol
6 Read the Percenl Transm 1

CALCULATION Of RESULTS A ot the Unknown1T11I1

X Value of tne XO 01 1l
Suandard
Value or the unknownttesl = A of the siandard
(mmoVLl

u- ,y 2111 Ed.)
REFERENCE INTERVAi. (nwn
SI un~
convenlloffal Unit a.11 -2 15 mmolll
Tngtyc:endd 10 _ 190 mgtdl
Serum(FllSflng)
42