Short Communication Agr. Bioi. Chem.

, 37 (4), 945~947, 1973

which have a more 'equatorial' C a substituent

Application ofAxial-haloketone were studied.
The structures of the lactones are shown
Rule to Lactones in Fig. 1. The non-bridged lactones were
prepared from D( - )-pantolactone I (a) and
Hiroshi MEGURO, Toshio KONNO L( + )-homoserine-r-lactone hydrochloride VII
and Katura TUZIMURA (b), and a,r-bridged iactones were from D( -)-
Department of Food Chemistry, Faculty of quinide XIII by the reactions which are
Agriculture, Tohoku University, Sendai, Japan known to proceed with the retention or the
Received October 28, 1972 conversion of the configuration, all having a
heteroatom attached to Ca. For comparison
In ketone formation of a strong and in- the CD of dihydrocoronopilin X/) r-tetrahy-
herently dissymmetric chromophore due to the drosantonin XF) and gibberellin (A9 methyl-
interaction between lone pair electrons of the ester XII 8 )) are adopted from literatures.
axial halogen substituents at C a associated with They have a methyl group at Ca and the ring
a strong molecular rotation and red shift at puckering increases from i3,r-cis-bridged X,
the extremum has been known as the axial- ig,r-trans-bridged XI to a,r-bridged lactones,
haloketone rule. I ) Although optical rotatory associated with increasing [8] and a red shift
dispersion (ORD) and circular dichroism (CD) at the CD maximum.
of many lactone have been reported,Z-S) the The molecular ellipticities [8], the wave
application of the rule to lactone has not yet length at the CD maximum and the red shift
~

been fully studied. However the skeleton of the maximum from X in non bridged
contribution of the lactone ring has been lactones and from XII in a,r-bridged lactones
studied both in 0_ 4 ) and r 5 '-lactones. are shown in Table I. The following should
In this communication we present the CD be noted in non-bridged lactones. (1) The
of some lactones which suggests the possi- sign of Cotton effect (C.E.) was determined
bility of applying of this rule to lactones. according to the configuration at Ca. The R
Two types of lactones, non-bridged r-iactones (or S) configuration gave positive (or negative)
1- IX which have a more 'axiaI'9) Ca sub- sign which agrees with the prediction from the
stituent and a,r-bridged lactones XII-XVII axial-haloketone rule when the rule is applied

CH
- ..l H
HO, / CH,)
H 0 /

0
H H , ,H
R ,
R H
R' H CH,

H' ,cCH, r I,I-IVJI

ia)
i x) ( Xl )
h H'H (VJI)-ilX)
CH,
R 0 -(J
H/'.,
,"+--- /
'I

o o
c=o
,
c=o ,"
,',/' c=o
,
1

R ,1
CH l COOCH 3
R
( Xll ) (XIII) R=OH
[XV). H=OH
,
) XN) R~O-C- CH 3
:i (XVJ· f-( - 1)'\"
o U,
,XVII !lOS-CH,
II
o
FIG. 1. The Structures of the Lactones Shown in Table 1.
946 H. MEGURO, T. KONNO and K. TUZIMURA

TABLE I. CD AND ORD, SIGN, MAGNITUDE AND RED SHIFT

CD and ORD measurements. On a JASCO ORDjUV-5, 1 cm cell, about 1 mgjml in methanol
except III in ether and IX in ethanol. [01, corrected relatively using D-pantolactone ([Oh22 = -12. 8 x 103
in 95 % ethanol) at 25°C. VIII and IX were partially racemized and should have qualitative values. XIII
and XV have also been reported 51 without the above-correction and qualitatively accorded with our's.
C-2 CD Max. ORD 1st. Ext.
Compo C.E.
•
sIgn [01 x 10- 3 (nm) Red shift [Ml x 10-3 (nm)
subst. config.
Non bridged from (X)
(a) (I) -OH R -12.5 (223) + 8 -5.7 (236)
(II) -OCH3 R - 6.9 (225) +10 -2.2 (240)
(III) -OSi(CH3h R 7.7 (226) +11 -1.6 (240)
(IV) -OCCH3 R - 6.6 (220) + 5 -3.0 (235)
II
0
(V) -OS02CH3 R 6.8 (221) + 6 -2.4 (235)
(VI) -Cl S + + 8.3 (220) + 5 +3.0 (237)
(b) (VII) -NH2 S + + 5.3 (217) + 2 +1.9 (234)
-NH+Cl-3 S + + 1.6 (221) + 5 -0.035 (238)
(VIII) -Cl R 5.4 (226) +11 -1.5 (247)
(IX) -Br R - 8.0 (232) +16 -2.1 (258)
p,T-Bridged
(X) -CH3 + + 3.5 (215)
(XI) -CH3 R + + 4.5 (217)
a,T-Bridged from(XII)
(XII) -CH3 R - 5.3 (222) 0
(XIII) -OH S - 8.9 (222) 0 -4.3 (236)
(XIV) -OCCH3 S -12.7 (217) 5 -6.0 (234)
0"
(XV) -OH S - 7.9 (222) 0 -4.5 (236)
(XVI) -OCCH3 S - -14.5 (218) ~ -6.4 (233)
II
0
(XVII) -OS02CH3 S -11.3 (220) 2 -5.2 (236)

to lactones. '1 On the other hand, in a,r-bridged lactones, the

(2) The heteroatoms gave a red shift of sign of C.E. was determined by the skeleton
about 5 -16 nm from the methyl group as contribution according to Beecham's rule 51
in the case of axial halogens in ketones. and the introduction of heteroatoms such as
The red shift was sensitive to the nature of hydroxyl, acetohydroxyl, and methanesulfonyl
substituents and also to a slight difference of groups instead of methyl groups XII gave
conformation. a negligibly small red shift or a rather small
(3) They gave larger [8] as compared with X blue shift as inthe case of equatorial halogens
and XI, suggesting that the contribution of the in ketones. •

C a 'axial' heteroatom might be large enough The red shift of CD maximum and contri-
to cancell the skeleton contribution. In the bution to the CD sign by 'axial' heteroatoms
two series of lactones (a) and (b) the order of in non-bridged r-Iactones and the small blue
the magnitude is as follows: shift and negligible contribution to the CD
• • • • • • • •
sign by 'equatorial' heteroatoms in a,r-bridged
(a) -OH>-CI: >-OSi(CH3h>-OS02CH3 lactones strongly suggest the possibility of
• • •• • • ••
• • •• applying of the axial-halo ketone rule to lac-
>-OCH3>-OCOCH3>6600.
•• •• tones. Here the substituents are not limited
• • • • •• to halogens but can be more widely eliiended
(b) -Br: >-NH2' -CI: >5700.
• • •• to other heteroatoms such as. o~ygen and
 Application ofAxial-haloketone Rule to Lactones 947

nitrogen so long as they have lone pair elec- REFERENCES

trons. 1) C. Djerassi, "Optical Rotatory Dispersion, Ap-
The formation of a inherently dissymmetric plication to Organic Chemistry," McGraw-Hill
chromophore between lactone carbonyl and Co. Inc., 1960; A. Moscowitz, K. Mislow, M. A.
heteroatoms was also shown in the solvent Glass and C. Djerassi, J. Am. Chem. Soc., 84,
effect of VII in which the amino group at Ca 1962 (1945).
2) W. Klyne, P. M. Scopes and A. Williams, J.
gave a very weak CD peak when the lone pair
Chem. Soc., 1965, 7237.
electrons of nitrogen were masked in the form 3) T. Okuda, S. Harigaya and A. Kiyomoto, Chem.
of hydrochloride. However, it increased to a Pharm. Bull. Japan, 12, 504 (1964).
strong band four times more than in a form of 4) H. Wolf, Tetrahedron Letters, 1965, 1075; 1966,
free amine in which the lone pair electrons of 5151.
5) A. F. Beecham, a. Terahedron Letters, 1968,
nitrogen were exposed after neutralization
2355; b. ibid., 1968, 3591; c. ibid., 1969, 4897.
with KOH. 6) H. Meguro, K. Hachiya, A. Tagiri and K. Tuzi-
mura, Agr. Bioi. Chem., 36, 2075 (1972).
We thank Kyowa Hakko Kogyo Co. and Daiichi
7) W. Stoklin, T. G. Waddell and T. A. Geissman,
Seiyaku Co. for the gift of their Dreiding models of
Tetrahedron, 26, 2397 (1970).
L-homoserine and D-pantolactone. Thanks are also
8) H. Meguro, K. Tuzimura and N. Takahasi,
due to Y. Sugiyama, T. Yamazaki for the preparation
Tetrahedron Letters, 1968, 6305.
·of some of the samples, and Mrs. A. Tagiri for the help
9) Here the terms, more 'axial' and more 'equatorial,'
.of CD measurements. are used tentatively to express the difference in
the projection angle of the substituents from the
lactone plane by inspection.
 Short Communication Agr. Bioi. Chem., 37 (4), 945~947, 1973

which have a more 'equatorial' C a substituent

Application ofAxial-haloketone were studied.
The structures of the lactones are shown
Rule to Lactones in Fig. 1. The non-bridged lactones were
prepared from D( - )-pantolactone I (a) and
Hiroshi MEGURO, Toshio KONNO L( + )-homoserine-r-lactone hydrochloride VII
and Katura TUZIMURA (b), and a,r-bridged iactones were from D( -)-
Department of Food Chemistry, Faculty of quinide XIII by the reactions which are
Agriculture, Tohoku University, Sendai, Japan known to proceed with the retention or the
Received October 28, 1972 conversion of the configuration, all having a
heteroatom attached to Ca. For comparison
In ketone formation of a strong and in- the CD of dihydrocoronopilin X/) r-tetrahy-
herently dissymmetric chromophore due to the drosantonin XF) and gibberellin (A9 methyl-
interaction between lone pair electrons of the ester XII 8 )) are adopted from literatures.
axial halogen substituents at C a associated with They have a methyl group at Ca and the ring
a strong molecular rotation and red shift at puckering increases from i3,r-cis-bridged X,
the extremum has been known as the axial- ig,r-trans-bridged XI to a,r-bridged lactones,
haloketone rule. I ) Although optical rotatory associated with increasing [8] and a red shift
dispersion (ORD) and circular dichroism (CD) at the CD maximum.
of many lactone have been reported,Z-S) the The molecular ellipticities [8], the wave
application of the rule to lactone has not yet length at the CD maximum and the red shift
~

been fully studied. However the skeleton of the maximum from X in non bridged
contribution of the lactone ring has been lactones and from XII in a,r-bridged lactones
studied both in 0_ 4 ) and r 5 '-lactones. are shown in Table I. The following should
In this communication we present the CD be noted in non-bridged lactones. (1) The
of some lactones which suggests the possi- sign of Cotton effect (C.E.) was determined
bility of applying of this rule to lactones. according to the configuration at Ca. The R
Two types of lactones, non-bridged r-iactones (or S) configuration gave positive (or negative)
1- IX which have a more 'axiaI'9) Ca sub- sign which agrees with the prediction from the
stituent and a,r-bridged lactones XII-XVII axial-haloketone rule when the rule is applied

CH
- ..l H
HO, / CH,)
H 0 /

0
H H , ,H
R ,
R H
R' H CH,

H' ,cCH, r I,I-IVJI

ia)
i x) ( Xl )
h H'H (VJI)-ilX)
CH,
R 0 -(J
H/'.,
,"+--- /
'I

o o
c=o
,
c=o ,"
,',/' c=o
,
1

R ,1
CH l COOCH 3
R
( Xll ) (XIII) R=OH
[XV). H=OH
,
) XN) R~O-C- CH 3
:i (XVJ· f-( - 1)'\"
o U,
,XVII !lOS-CH,
II
o
FIG. 1. The Structures of the Lactones Shown in Table 1.
946 H. MEGURO, T. KONNO and K. TUZIMURA

TABLE I. CD AND ORD, SIGN, MAGNITUDE AND RED SHIFT

CD and ORD measurements. On a JASCO ORDjUV-5, 1 cm cell, about 1 mgjml in methanol
except III in ether and IX in ethanol. [01, corrected relatively using D-pantolactone ([Oh22 = -12. 8 x 103
in 95 % ethanol) at 25°C. VIII and IX were partially racemized and should have qualitative values. XIII
and XV have also been reported 51 without the above-correction and qualitatively accorded with our's.
C-2 CD Max. ORD 1st. Ext.
Compo C.E.
•
sIgn [01 x 10- 3 (nm) Red shift [Ml x 10-3 (nm)
subst. config.
Non bridged from (X)
(a) (I) -OH R -12.5 (223) + 8 -5.7 (236)
(II) -OCH3 R - 6.9 (225) +10 -2.2 (240)
(III) -OSi(CH3h R 7.7 (226) +11 -1.6 (240)
(IV) -OCCH3 R - 6.6 (220) + 5 -3.0 (235)
II
0
(V) -OS02CH3 R 6.8 (221) + 6 -2.4 (235)
(VI) -Cl S + + 8.3 (220) + 5 +3.0 (237)
(b) (VII) -NH2 S + + 5.3 (217) + 2 +1.9 (234)
-NH+Cl-3 S + + 1.6 (221) + 5 -0.035 (238)
(VIII) -Cl R 5.4 (226) +11 -1.5 (247)
(IX) -Br R - 8.0 (232) +16 -2.1 (258)
p,T-Bridged
(X) -CH3 + + 3.5 (215)
(XI) -CH3 R + + 4.5 (217)
a,T-Bridged from(XII)
(XII) -CH3 R - 5.3 (222) 0
(XIII) -OH S - 8.9 (222) 0 -4.3 (236)
(XIV) -OCCH3 S -12.7 (217) 5 -6.0 (234)
0"
(XV) -OH S - 7.9 (222) 0 -4.5 (236)
(XVI) -OCCH3 S - -14.5 (218) ~ -6.4 (233)
II
0
(XVII) -OS02CH3 S -11.3 (220) 2 -5.2 (236)

to lactones. '1 On the other hand, in a,r-bridged lactones, the

(2) The heteroatoms gave a red shift of sign of C.E. was determined by the skeleton
about 5 -16 nm from the methyl group as contribution according to Beecham's rule 51
in the case of axial halogens in ketones. and the introduction of heteroatoms such as
The red shift was sensitive to the nature of hydroxyl, acetohydroxyl, and methanesulfonyl
substituents and also to a slight difference of groups instead of methyl groups XII gave
conformation. a negligibly small red shift or a rather small
(3) They gave larger [8] as compared with X blue shift as inthe case of equatorial halogens
and XI, suggesting that the contribution of the in ketones. •

C a 'axial' heteroatom might be large enough The red shift of CD maximum and contri-
to cancell the skeleton contribution. In the bution to the CD sign by 'axial' heteroatoms
two series of lactones (a) and (b) the order of in non-bridged r-Iactones and the small blue
the magnitude is as follows: shift and negligible contribution to the CD
• • • • • • • •
sign by 'equatorial' heteroatoms in a,r-bridged
(a) -OH>-CI: >-OSi(CH3h>-OS02CH3 lactones strongly suggest the possibility of
• • •• • • ••
• • •• applying of the axial-halo ketone rule to lac-
>-OCH3>-OCOCH3>6600.
•• •• tones. Here the substituents are not limited
• • • • •• to halogens but can be more widely eliiended
(b) -Br: >-NH2' -CI: >5700.
• • •• to other heteroatoms such as. o~ygen and
 Application ofAxial-haloketone Rule to Lactones 947

nitrogen so long as they have lone pair elec- REFERENCES

trons. 1) C. Djerassi, "Optical Rotatory Dispersion, Ap-
The formation of a inherently dissymmetric plication to Organic Chemistry," McGraw-Hill
chromophore between lactone carbonyl and Co. Inc., 1960; A. Moscowitz, K. Mislow, M. A.
heteroatoms was also shown in the solvent Glass and C. Djerassi, J. Am. Chem. Soc., 84,
effect of VII in which the amino group at Ca 1962 (1945).
2) W. Klyne, P. M. Scopes and A. Williams, J.
gave a very weak CD peak when the lone pair
Chem. Soc., 1965, 7237.
electrons of nitrogen were masked in the form 3) T. Okuda, S. Harigaya and A. Kiyomoto, Chem.
of hydrochloride. However, it increased to a Pharm. Bull. Japan, 12, 504 (1964).
strong band four times more than in a form of 4) H. Wolf, Tetrahedron Letters, 1965, 1075; 1966,
free amine in which the lone pair electrons of 5151.
5) A. F. Beecham, a. Terahedron Letters, 1968,
nitrogen were exposed after neutralization
2355; b. ibid., 1968, 3591; c. ibid., 1969, 4897.
with KOH. 6) H. Meguro, K. Hachiya, A. Tagiri and K. Tuzi-
mura, Agr. Bioi. Chem., 36, 2075 (1972).
We thank Kyowa Hakko Kogyo Co. and Daiichi
7) W. Stoklin, T. G. Waddell and T. A. Geissman,
Seiyaku Co. for the gift of their Dreiding models of
Tetrahedron, 26, 2397 (1970).
L-homoserine and D-pantolactone. Thanks are also
8) H. Meguro, K. Tuzimura and N. Takahasi,
due to Y. Sugiyama, T. Yamazaki for the preparation
Tetrahedron Letters, 1968, 6305.
·of some of the samples, and Mrs. A. Tagiri for the help
9) Here the terms, more 'axial' and more 'equatorial,'
.of CD measurements. are used tentatively to express the difference in
the projection angle of the substituents from the
lactone plane by inspection.
Wh
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 PAPER CHROMATOGRAPHY

INTRODUCTION

Chromatography is a technique that is used to separate and to identify components of a

mixture. This analytical technique has a wide range of applications in the real world since
many substances are mixtures of chemical compounds. In this lab, you will explore two
applications of chromatography - identification of an unknown ink sample and the separation of
food colorings.

In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of
the paper is immersed in a solvent, and the solvent moves up the paper by capillary action.
Components of the mixture are carried along with the solvent up the paper to varying degrees,
depending on the compound's preference to be adsorbed onto the paper versus being carried
along with the solvent. The paper is composed of cellulose to which polar water molecules are
adsorbed, while the solvent is less polar, usually consisting of a mixture of water and an
organic liquid. The paper is called the stationary phase while the solvent is referred to as the
mobile phase. Performing a chromatographic experiment is basically a three-step process: 1)
application of the sample, 2) "developing" the chromatogram by allowing the mobile phase to
move up the paper, and 3) calculating Rf values and making conclusions.

In order to obtain a measure of the extent of movement of a component in a paper

chromatography experiment, we can calculate an "Rf value" for each separated
component in the developed chromatogram. An Rf value is a number that is defined as:

distance traveled by component from application point

Rf = ----------------------------------------------------
distance traveled by solvent from application point

The distance traveled by the spot is measured to the MIDDLE of the spot.

Solvent Front

B Rf = A/B
A

Pencil Mark
Forensic chemistry, the profession popularized by TV's "Quincy" is the application of
chemical techniques to law. Many samples that are received in a crime laboratory are
complex mixtures of chemical compounds and chromatography is often ideal for
separating and identifying these components. One example of this is the identification
of an unknown ink sample. Suppose the authenticity of a signature or the source of a
ransom note is in question. Using paper chromatography, it is often possible to identify
the source of the inks that were used and thereby provide strong evidence that can be
used in the courtroom

In the second part of the experiment, you will separate a mixture of three food colorings
using "reverse-phase" chromatography. In this type of chromatography, the stationary
phase is a non-polar material and the mobile phase is relatively polar. You will use a
commercial C18 cartridge, which contains very non-polar 18-carbon long chains as the
stationary phase. The mobile phase will be water and/or isopropyl alcohol (IPA).

TECHNIQUES Using chromatography paper strips

AVOID EXCESSIVE HANDLING OF PAPER

1. The chromatography paper is cut in about 2.5 x 10 cm strips. Along one of the
shorter sides, draw a horizontal line in pencil (lead will not move) about 1.5 cm
from the edge of the strip. This will be your “base line”, the starting line where
the samples will be spotted.

2. Apply a dot of ink from each black felt tip pen on the baseline. Make sure dots
are evenly spaced on the baseline and not too close to the edge. It may be
helpful to use a pencil to mark the spots before placing the ink on the paper. See
the diagram below.

3. Label each spot in pencil so that you know what each spot is.

4. Stand the paper in the eluting solution. Taping the top of the strips to the side of
the beaker may be helpful.

B a s e L in e
1 2 3
Using C18 cartridge

1. Remove the cartridge from the bottom of the syringe then remove the piston of
the syringe.

2. Insert the LONGER END of the C18 cartridge snugly onto the syringe tip. Be
careful to use the proper end and not put the cartridge on backwards.

3. Add solution as directed and insert the piston.

4. Flush solutions slowly through the cartridge. Faster is not better.

5. REMOVE CARTRIDGE BEFORE PULLING PLUNGER to add new solution to

avoid pulling air backwards through the column.

6. The cartridge should be cleaned with a 1:1 mixture of water:IPA until all coloring
is removed. Food coloring not removed may stain the column and make it less
effective.

SAFETY AND DISPOSAL

• The food coloring mixture will stain your skin and clothing. Handle with care.
• All of the solutions in this lab may be washed down the drain with water.

EXPERIMENTAL PROCEDURE

I. Identification of Inks by Paper Chromatography

A. This lab will be done as a collaboration between both lab pairs at a bench.
1. One lab pair will use 2 parts isopropanol to 1 part water as a solvent
(labeled 2:1 IPA).
2. One lab pair will use 1 part isopropanol to 2 parts water as a solvent
(labeled 1:2 IPA).
B. Obtain a strip of chromatography paper about 2.5 cm wide by 10 cm tall.
Along one of the shorter sides, draw a horizontal line in pencil about
1.5 cm from the edge of the strip. This will be your “base line”, the
starting line where the samples will be spotted. Graphite will not be
carried up the chromatography paper. This baseline will be used later in
the calculations of Rf values.
C. Using the black felt tip pens, apply generous dots of ink on the baseline
according to the diagram in the "TECHNIQUES" section.
D. With a pencil, label each dot with the identity of the pen from which the ink came.
E. Obtain a paper strip from your instructor that is marked with one of the three
pens. This is the unknown ink sample that you must identify.
F. Once your two strips are prepared, set up a developing chamber using a 400 ml
beaker.
 1. Pour some of the developing solvent in the beaker, using only enough solvent
to cover the bottom of the beaker (about 5-10 mL).
2. Remember, one lab pair will use 1:2 IPA and the other lab pair will use 2:1
IPA.
G. Place the paper strips into the developing chamber so that the edge near the ink
spots is submerged in the solvent. THE INK SPOT MUST BE ABOVE THE
SOLVENT LEVEL. You should avoid allowing the paper strips to touch each
other or the sides of the beaker. (Taping the top of the strips to the outside of the
beaker may be helpful.) Once you have inserted the paper, cover the chamber
with a watchglass and make sure the solvent is progressing up the paper.
H. When the solvent has risen almost to the top of the paper strip, remove the strip
from the chamber and immediately mark the level to which the solvent has risen
and circle each spot present on the strip with a pencil. (The solvent front and the
spots will continue to move for several minutes after removing the strip from the
beaker.)
I. Measure the distance each spot traveled and calculate Rf values for each spot.
Use the circles you drew around each spot with your pencil and not the actual
spot!
J. Turn your chromatogram in as part of your report.

II. Separation of Food Colorings

A. Remove the piston and insert the LONG END of the C18 cartridge snugly
into the syringe tip.
B. Add 5 mL of IPA, insert the piston, and slowly flush at a flow rate not
exceeding 5-10 mL per minute. It is important to not flush liquid through
the cartridge more rapidly than 10 mL per minute. Repeat this step with
another 5 mL portion of IPA.
C. Flush with two 5 mL portions of distilled water.
D. Add about 1 mL of the food coloring mixture to the syringe, insert the
piston, and push the mixture onto the cartridge. As you push the sample
through the cartridge, immediately begin collecting the drops in a test
tube. The first color comes off quickly.
1. Once the 1 mL of food coloring mixture is pushed onto the
cartridge, add water to the syringe and push it through the cartridge
until all the yellow is gone. Collect the yellow liquid. The process
of pushing solvent(s) through a cartridge is called elution. In this
case, water elutes the yellow color.
2. The remaining colors in the mixture must be removed by eluting
with a water/IPA mixture. Prepare a 1% IPA solution by placing 5
mL of water into the syringe and adding 1 drop of IPA. Mix well by
shaking the syringe. Push this solution through the cartridge and
collect all the colors that come off. If coloring still remains on the
cartridge, prepare a 2% IPA solution by adding 2 drops of IPA to 5
mL of water, mix, and elute this solution through the cartridge.
Continue to increase the percentage of IPA (3% - 3 drops IPA + 5
mL water, 4% - 4 drops IPA + 5 mL water, etc.) until all or most of
the coloring has been removed from the cartridge. A 5% IPA
 solution may be the most concentrated solution needed to remove
all of the colors from the cartridge.
3. Any coloring residue remaining on the cartridge should be removed
using a 1:1 IPA:water mixture. This residue may need to be
collected.
4. Save the separated colored solutions for the next part.

E. See what would happen if several of the separated colored solutions were
mixed.
- Try to make a green solution from the colors you separated.

F. Make sure the cartridge is clean before returning it.

 ISSN 0975 – 8542
Journal of Global Pharma Technology
Available Online at [Link]
REVIEW ARTICLE

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY: A SHORT

REVIEW

Malviya R, Bansal V*, Pal O.P. and Sharma P.K.

Department of pharmaceutical technology, Meerut Institute of Engineering and Technology,

Meerut, India
*For correspondence: Email: vipinbansal1986@[Link]

Abstract: High performance liquid chromatography (HPLC) is an important qualitative and quantitative
technique, generally used for the estimation of pharmaceutical and biological samples. It is the most
versatile, safest, dependable and fastest chromatographic technique for the quality control of drug
components. This article was prepared with an aim to review different aspects of HPLC, such as principle,
types, instrumentation and application.

Keywords: High performance liquid chromatography, instrumentation, elution, applications, mobile phase.

INTRODUCTION miscible combinations of water or organic

liquids (the most common are methanol
High-performance liquid chromatography and acetonitrile). [2, 3] Sepration has been
(or High pressure liquid chromatography, done to vary the mobile phase composition
HPLC) is a specific form of column during the analysis; this is known as
chromatography generally used in gradient elution. [3] The gradient separates
biochemistry and analysis to separate, the analyte mixtures as a function of the
identify, and quantify the active affinity of the analyte for the current
compounds. [1] HPLC mainly utilizes a mobile phase. The choice of solvents,
column that holds packing material additives and gradient depend on the
(stationary phase), a pump that moves the nature of the stationary phase and the
mobile phase(s) through the column, and a analyte.
detector that shows the retention times of
the molecules. Retention time varies TYPES OF HPLC
depending on the interactions between the
stationary phase, the molecules being Types of HPLC generally depend on phase
analyzed, and the solvent(s) used. [2] The system used in the process. [3, 4]
sample to be analyzed is introduced in Following types of HPLC generally used
small volume to the stream of mobile in analysis-
phase and is retarded by specific chemical
or physical interactions with the stationary Normal phase chromatography: Also
phase. The amount of retardation depends known Normal phase HPLC (NP-HPLC),
on the nature of the analyte and this method separates analytes based on
composition of both stationary and mobile polarity. NP-HPLC uses a polar stationary
phase. The time at which a specific analyte phase and a non-polar mobile phase. The
elutes (comes out of the end of the polar analyte interacted with and is
column) is called the retention time. retained by the polar stationary phase.
Common solvents used include any Adsorption strengths increase with
© 2009, JGPT. All Rights Reserved. 22
 Bansal V. et al., Journal of Global Pharma Technology. 2010; 2(5): 22-26

increased analyte polarity, and the Proteins bound to a bioaffinity column can
interaction between the polar analyte and be eluted in two ways:
the polar stationary phase increases the • Biospecific elution: inclusion of free
elution time. ligand in elution buffer which
competes with column bound ligand.
Reversed phase chromatography: • Aspecific elution: change in pH, salt,
Reversed phase HPLC (RP-HPLC or RPC) etc. which weakens interaction protein
has a non-polar stationary phase and an with column-bound substrate.
aqueous, moderately polar mobile phase. Because of specificity of the interaction,
RPC operates on the principle of bioaffinity chromatography can result in
hydrophobic interactions, which result very high purification in a single step (10 -
from repulsive forces between a polar 1000-fold).
eluent, the relatively non-polar analyte,
and the non-polar stationary phase. The PARAMETERS
binding of the analyte to the stationary
phase is proportional to the contact surface For the accurate analysis of a compound,
area around the non-polar segment of the there are some parameters which are used
analyte molecule upon association with the as a standard for a particular compound. If
ligand in the aqueous eluent. there is a change occurs in the parameters
the result may be affected greatly. The
Size exclusion chromatography: Size most commonly used parameters are
exclusion chromatography (SEC), also internal diameter, particle size, pore size,
called as gel permeation chromatography pump pressure. For different compounds
or gel filtration chromatography mainly the parameters can be changed according
separates particles on the basis of size. It is to their nature and chemical properties.
also useful for determining the tertiary
structure and quaternary structure of Internal diameter: The internal diameter
proteins and amino acids. This technique is (ID) of an HPLC column is a critical
widely used for the molecular weight aspect that determines quantity of analyte
determination of polysaccharides. that can be loaded onto the column and
also influences sensitivity. Larger columns
Ion exchange chromatography: In Ion- are usually seen in industrial applications
exchange chromatography, retention is such as the purification of a drug product
based on the attraction between solute ions for later use. Low ID columns have
and charged sites bound to the stationary improved sensitivity and lower solvent
phase. Ions of the same charge are consumption at the expense of loading
excluded. This form of chromatography is capacity.
widely used in purifying water, Ligand-
exchange chromatography, Ion-exchange Particle size: Most traditional HPLC is
chromatography of proteins, High-pH performed with the stationary phase
anion-exchange chromatography of attached to the outside of small spherical
carbohydrates and oligosaccharides, etc. silica particles (very small beads). Smaller
[3, 4] particles generally provide more surface
area and better separations, but the
Bio-affinity chromatography: Separation pressure required for optimum linear
based on specific reversible interaction of velocity increases by the inverse of the
proteins with ligands. Ligands are particle diameter squared.
covalently attached to solid support on a
bio-affinity matrix, retains proteins with Pore size: Many stationary phases are
interaction to the column-bound ligands. porous to provide greater surface area.

© 2009, JGPT. All Rights Reserved. 23

 Bansal V. et al., Journal of Global Pharma Technology. 2010; 2(5): 22-26

Small pores provide greater surface area calculated automatically by the computer
while larger pore size has better kinetics linked to the display.
especially for larger analytes. Pore size
defines an ability of the analyte molecules APPLICATION
to penetrate inside the particle and interact
with its inner surface. This is especially The information that can be obtained using
important because the ratio of the outer HPLC includes identification,
particle surface to its inner one is about quantification, and resolution of a
1:1000. The surface molecular interaction compound. Preparative HPLC refers to the
mainly occurs on the inner particle surface. process of isolation and purification of
compounds. This differs from analytical
Pump pressure: Pumps vary in pressure HPLC, where the focus is to obtain
capacity, but their performance is information about the sample compound.
measured on their ability to yield a
consistent and reproducible flow rate. Chemical Separations It is based on the
Modern HPLC systems have been fact that certain compounds have different
improved to work at much higher migration rates given a particular column
pressures, and therefore be able to use and mobile phase, the extent or degree of
much smaller particle sizes in the columns separation is mostly determined by the
(< 2 micrometres). choice of stationary phase and mobile
phase.
INSTRUMENTATION
Purification: Purification is defined as the
Injection of the sample: Septum injectors process of separating or extracting the
are available; using which sample solution target compound from a mixture of
is injected. Sample can be injected when compounds or contaminants. Each
the mobile phase is flowing or it is compound showed a characteristic peak
stopped. A new advanced rotary valve and under certain chromatographic conditions.
loop injector can be used to produce The migration of the compounds and
reproducible results. contaminants through the column need to
differ enough so that the pure desired
The detector: There are several ways of compound can be collected or extracted
detecting when a substance has passed without incurring any other undesired
through the column. Generally UV compound.
spectroscopy is attached, which detect the
specific compounds. Many organic Identification Generally assay of
compounds absorb UV light of various compounds are carried using HPLC. The
wavelengths. The amount of light parameters of this assay should be such
absorbed will depend on the amount of a that a clean peak of the known sample is
particular compound that is passing observed from the chromatograph. The
through the beam at the time. identifying peak should have a reasonable
retention time and should be well
Interpreting the output from the separated from extraneous peaks at the
detector: The output is recorded as a detection levels which the assay will be
series of peaks, each one representing a performed.
compound in the mixture passing through
the detector and absorbing UV light. The Other applications of HPLC: Other
area under the peak is proportional to the applications of HPLC includes
amount of substance, which is passed
through detector, and this area can be

© 2009, JGPT. All Rights Reserved. 24

 Bansal V. et al., Journal of Global Pharma Technology. 2010; 2(5): 22-26

Pharmaceutical applications [5-8] CONCLUSION

• Tablet dissolution study of
armaceutical dosages form. It can be concluded from the entire review
• Shelf-life determinations of that HPLC is a versatile, reproducible
parmaceutical products chromatographic technique for the
• Identification of active ingredients of estimation of drug products. It has wide
dosage forms applications in different fields in term of
• Pharmaceutical quality control quantitative and qualitative estimation of
active molecules.
Environmental applications [9-12]
• Detection of phenolic compounds in REFERENCES
Drinking Water
1. Martin M., Guiochon, G. Effects of
• Identification of diphenhydramine in high pressures in liquid
sedimented samples chromatography. J. Chromatogr. A,
• Bio-monitoring of pollutant 2005; (1-2)7: 16-38.

Forensics [13-15] 2. Liu Y., Lee M.L. Ultrahigh pressure

• Quantification of the drug in biological liquid chromatography using elevated
temperature. Journal of
samples. Chromatography. 2006; 1104 (1-2):
• Identification of anabolic steroids in 198–202.
serum, urine, sweat, and hair
• Forensic analysis of textile dyes. 3. Abidi,S.L. High-performance liquid
• Determination of cocaine and chromatography of phosphatidic acids
and related polar lipids. J. Chromatogr.
metabolites in blood 1991; 587: 193-203.
Clinical [16-19] 4. Hearn M.T.W. Ion-pair
• Quantification of ions in human urine chromatography on normal and
Analysis of antibiotics in blood reversed-phase systems. Adv.
plasma. Chromatogr. 1980; 18: 59–100.
• Estimation of bilirubin and bilivirdin in 5. Bergh J. J., Breytenbach, J. C.
blood plasma in case of hepatic Stability-indicating High-performance
disorders. Liquid- chromatographic Analysis of
• Detection of endogenous Trimethoprim in Pharmaceuticals. J.
neuropeptides in extracellular fluids of Chromatogr. 1987; 387: 528-531.
brain.
6. Stubbs C., Kanfer, I. Stability-indi-
cating High-performance Liquid-
FoodandFlavor [20] chromato- graphic Assay of
• Ensuring the quality of soft drink and Erythromycin Estolate in
drinking water. Pharmaceutical Dosage Forms. Int. J.
• Analysis of beer. Pharm. 1990; 3(2): 113-119.
• Sugar analysis in fruit juices. 7. MacNeil L., Rice J. J., Muhammad N.
• Analysis of polycyclic compounds in Lauback R. G. Stability-indicating
vegetables. Liquid-chromatographic Determination
• Trace analysis of military high of Cefapirin, Desacetylcefapirin and
explosives in agricultural crops. Cefapirin Lactone in Sodium Cefapirin
Bulk and Injectable Formulations. J.
Chromatogr. 1986; 361: 285-290.
8. Bounine J. P., Tardif B., Beltran P.
Mazzo D. J. High-performance Liquid-

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chromatographic Stability-indicating tissue. Antimicrob. Agents Chemother.

Determination of Zopiclone in Tablets. 1986; 30: 31-233.
J. Chromatogr. 1994; 677(1): 87-93.
15. Haginaka J., Yasuda H., Uno T.,
9. Lauback R. G., Rice J. J., Bleiberg B., Nkagawa T. Alkaline degradation and
Muhammad N., Hanna, S. A. 1984. determination by high-performance by
Specific High-performance Liquid- high-performance liquid
chromato- graphic Determination of chromatography. Chem. Pharm. Bull.
Ampicillin in Bulks, Injectables, 1984; 32: 2752-2758
Capsules and Oral Suspensions by
Reversed-phase Ion-pair 16. Fredj G., Paillet Aussel M. F., Brouard
Chromatography. J. Liq. Chromatogr. A., Barreteau H., Divine C., Micaud
1984; 7(6): 1243-1265. M. Determination of sulbactam in
biological fluids by high-performance
10. Wiklund A E., Dag B., Brita S. liquid chromatography. J. Chromatogr.
Toxicity evaluation by using intact 1986; 383: 218-222.
sediments and sediment extracts.
Marine Pollution Bulletin (2005); 17. Rodenas V., Garcia M.S., Sanchez-
50(6): 660-667. Pedreno C., Albero M.I. Flow-
injection spectrophotometric
11. Kwok Y. C., Hsieh D. P. H., Wong P. determination of frusemide or
K. Toxicity identification evaluation sulphathiazole in pharmaceuticals. J.
(TIE) of pore water of contaminated Pharm. Biomed. Anal. 1997; 15: 1687-
marine sediments collected from Hong 1693.
Kong waters. Marine Pollution
Bulletin. 2005; 51(8-12): 1085-1091. 18. Shah A.J., Adlard M.W., Stride J.D. A
sensitive assay for clavulanic acid and
12. Hongxia Yu., Jing C., Cui Y., Shang sulbactam in biological fluids by high-
H., Ding Z., Jin H. Application of performance liquid chromatography
toxicity identification evaluation and precolumn derivatization. J.
procedures on wastewaters and sludge Pharm. Biomed. Anal. 1990; 5: 437-
from a municipal sewage treatment 443.
works with industrial inputs.
Ecotoxicology and Environmental 19. Abidi S.L. High-performance liquid
Safety. 2004; 57(3): 426-430. chromatography of phosphatidic acids
and related polar lipids. [Link].
13. Ayerton J. Assay of ceftazidime in 1991; 587: 193-203.
biological fluids using high-pressure
liquid chromatography. J. Antimicrob. 20. Christie W.W., Gill S., Nordbäck J.,
Chemother. 1981; 8: 227-231. Itabashi Y., Sanda S., Slabas A.R. New
procedures for rapid screening of leaf
14. Bowden R.E., Madsen P.O. High- lipid components from Arabidopsis.
pressure liquid chromatographic assay Phytochemical Anal. 1998; 9: 53-57.
of sulbactam in plasma, urine and

© 2009, JGPT. All Rights Reserved. 26

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Thin Layer Chromatography.

Thin layer chromatography, or TLC, is a method for analyzing mixtures by separating the
compounds in the mixture. TLC can be used to help determine the number of components in a
mixture, the identity of compounds, and the purity of a compound. By observing the appearance of
a product or the disappearance of a reactant, it can also be used to monitor the progress of a
reaction. TLC is a sensitive technique - microgram (0.000001 g) quantities can be analyzed by TLC
- and it takes little time for an analysis (about 5-10 minutes).

TLC consists of three steps - spotting, development, and visualization. Photographs of each step are
shown on the course website. First the sample to be analyzed is dissolved in a volatile (easily
evaporated) solvent to produce a very dilute (about 1%) solution. Spotting consists of using a micro
pipet to transfer a small amount of this dilute solution to one end of a TLC plate, in this case a thin
layer of powdered silica gel that has been coated onto a plastic sheet. The spotting solvent quickly
evaporates and leaves behind a small spot of the material.

Development consists of placing the bottom of the TLC plate into a shallow pool of a development
solvent, which then travels up the plate by capillary action. As the solvent travels up the plate, it
moves over the original spot. A competition is set up between the silica gel plate and the
development solvent for the spotted material. The very polar silica gel tries to hold the spot in its
original place and the solvent tries to move the spot along with it as it travels up the plate. The
outcome depends upon a balance among three polarities - that of the plate, the development solvent
and the spot material. If the development solvent is polar enough, the spot will move some distance
from its original location. Different components in the original spot, having different polarities, will
move different distances from the original spot location and show up as separate spots. When the
solvent has traveled almost to the top of the plate, the plate is removed, the solvent front marked
with a pencil, and the solvent allowed to evaporate.

Visualization of colored compounds is simple – the spots can be directly observed after
development. Because most compounds are colorless however, a visualization method is needed.
The silica gel on the TLC plate is impregnated with a fluorescent material that glows under
ultraviolet (UV) light. A spot will interfere with the fluorescence and appear as a dark spot on a
glowing background. While under the UV light, the spots can be outlined with a pencil to mark their
locations. A second method of visualization is accomplished by placing the plate into iodine vapors
for a few minutes. Most organic compounds will form a dark-colored complex with iodine. It is
good practice to use at least two visualization techniques in case a compound does not show up with
one particular method.

The Rf value is used to quantify the movement of the materials along the plate. Rf is equal to the
distance traveled by the substance divided by the distance traveled by the solvent. Its value is
always between zero and one. A TLC analysis might be summarized something like, "Using a silica
gel plate and ethyl acetate as the development solvent, unknown mixture X showed three spots
having Rf's of 0.12, 0.25, and 0.87". Comparing these Rf's with the Rf's of known compounds might
enable a tentative identification to be made. Note that observing three spots means only that there
are at least three components in the mixture. Some components may have such similar polarities
that they appear under one spot after development.

1
 Rf = Y/X (always ≤ 1)

If a development solvent of too high a polarity is used, all components in the mixture will move
along with the solvent and no separation will be observed (Rf’s will be too large). If the solvent is
of too low a polarity the components will not move enough, and again separation will not occur
(Rf’s will be too small). In practice, different solvents or mixtures of solvents are tried until a good
separation is observed. Typically an effective solvent is one that gives Rf's in the range of 0.3 - 0.7.

Note that the spotting solvent is simply used as a vehicle to transfer the material to be analyzed to
the TLC plate. Once the transfer is made the spotting solvent evaporates. It has no effect on the
separation. It is the development solvent that effects the separation.

What’s going on at the molecular level during development? There are three components in TLC:
(1) the TLC plate (stationary phase), the development solvent (mobile phase), and the sample to be
analyzed (solute). In our experiment the TLC plate consists of a thin plastic sheet covered with a
thin layer of silica gel, a portion of the structure of which is shown below.
OH OH OH

Si O Si O Si O

O O O

Si Si Si O

O O
Silica gel consists of a three-dimensional network of thousands of alternating silicon and oxygen
bonds, with O-H groups on the outside surface. Silica gel is simply very finely ground very pure
sand. It should be noted that silica gel is highly polar and is capable of hydrogen bonding.

Consider the side-on view of the development of a TLC plate below. As the solvent travels up the
plate, over the spot, an equilibrium is set up, as development solvent competes with the TLC plate
for the solute. The silica gel binds to the solute and the development solvent tries to dissolve it
away, carrying the solute(s) along as the solvent travels up the plate.

2
A balance of intermolecular forces determines the position of equilibrium and thus the ability of the
solvent to move the solute up the plate. In other words, would the spot prefer to be stuck on the
plate or would it prefer to move along with solvent.

The balance depends upon (1) the polarity of the TLC plate (constant and high), (2) the polarity of
the development solvent (can be varied by using different solvents), and (3) the polarity of the
compounds in the spot (this varies depending upon what compounds are in the spot). For example,
if a sample consists of two components, one more polar than the other, the more polar will tend to
stick more tightly to the plate and the less polar will tend to move along more freely with the
solvent. Using a more polar development solvent would cause both to move along further. If the
approximate structures of the solutes are known, it is possible to make an educated guess as to what
solvent or mixture of solvents to use. In practice though, for a given mixture of compounds to be
analyzed, a solvent or mixture of solvents is chosen by trial and error to give the best separation. (A
caveat: the polarity argument is helpful in understanding the principles of TLC. Because most
compounds have some polarity the argument works well. For compounds having very low polarity
however, a lower-polarity solvent may be more effective in moving the solute up the plate.)

To illustrate a TLC experiment, consider the following example of the analysis of a two-component
mixture.

3
The polarity of molecules, solutes and solvents alike, is ordered as follows, from least to most polar:

Alkanes (least polar), alkyl halides, alkenes, aromatic hydrocarbons, ethers, esters, ketones,
aldehydes, amines, alcohols, and carboxylic acids (most polar). Note however that many molecules
contain multiple functional groups and that the overall polarity would be determined by all of the
groups.

Experimental Procedure. (rev 1/08)

This experiment consists of two parts: (1) analyzing two different analgesics to determine the
number of compounds in each and the identity of those compounds and (2) studying the effect of
solvent polarity on Rf. Look over the photos showing TLC on the course website.

(1.) TLC analysis of analgesics.

Spotting. The powdered side of the silica gel plate, not the shiny, plastic side is the side that is
spotted. Lightly draw a pencil line about 1 cm from the end of a plate. Use a pencil, not a pen, and
be careful to not scratch the surface of the plate too deeply. The plates are of such a size that only
three spots can be run on each. Use a fresh pipet for each spot. Make each spot as small as possible
(less than about 1 mm diameter). After applying the spots check them under the UV lamp to see
that they are of a reasonable size. The UV light must be held close to the plate to see the spots
(CAUTION: do not look directly into the UV light.) If the spot is much too large, prepare a new
plate. If a spot is too small, add more solution. Analyze two unknown analgesics in the following
way: two of the three known compounds (references) plus the first analgesic to be analyzed
(unknown 1) will be spotted on one plate, the same two knowns plus the second analgesic
(unknown 2) will be spotted on a second plate, and both analgesic 1 and analgesic 2 along with the
third known compound will be spotted on a third plate (see Figure below). (If the plates were

4
wider, you could analyze each unknown on just one plate.) To obtain a sample of a known solution,
bring a micropipet to the sample solution at the side bench, dip it into the solution to fill it, then take
it back to your workspace for spotting (Use only micropipets for obtaining samples; do not use
Pasteur pipets to take larger amounts of samples – that could lead to contamination). Try to
minimize traffic at the side bench and take care to not contaminate the solutions with a micropipet
that has already been used. The purpose of the experiment is to determine which of the known
compounds are contained in the analgesics that you chose to analyze. Solutions of the known
compounds will already be made. You must make 1% solutions of the unknown analgesics. To do
this, take a small part of a crushed tablet and add enough ethanol to make an approximately 1%
solution (very approximate - do not weigh samples). About 1 mg in several drops of solvent will
produce an approximately 1% solution. The analgesic contains insoluble binders so not all of it will
dissolve.

Development. The development chamber is a small screw-capped jar with a 5.5 cm filter paper
placed into it to ensure that the atmosphere is saturated with vapor. Each run is relatively brief so
please use only one ethyl acetate development chamber. Otherwise there may not be enough for
everyone. Place about 2 mL of ethyl acetate into the chamber (2 mL is a full squeeze of the bulb
using a Pasteur pipet). Place the TLC plate into the jar with the spotted side at the bottom. Keep
the cap on the jar at all times except when placing the plate into or taking the plate out of the jar.
Watch out for the following: the solvent must be below the level of the spots or the spotted material
will dissolve in the development solvent; the plate must stand vertically in the tank and the silica gel
must not touch the filter paper; the solvent must not be allowed to run all of the way to the top of
the plate; the correct amount of material must be spotted (checking under UV light before
development may help - if spots are too large, another plate can be made - if the spots are too small,
more material can be spotted). The development takes little time so if a plate comes out poorly,
another can easily be done. After the solvent has almost reached the top of the plate, the plate is
removed and the position of the solvent front marked before the solvent evaporates. The plate is
then allowed to dry in the hood. Always allow the developing solvent to run almost completely to
the top of the plate. This will use the entire plate and allow for the best possible separation.

Visualization. This is done by two methods. First, by short wavelenth UV light. (CAUTION: do
not look directly into the light.) The light must be held close to the plate to see the the spots. Some
spots may be very faint. The observed spots should be outlined with a pencil. The plate is then
placed into a jar containing iodine crystals for a few minutes. Keep the iodine jar capped and in the
fume hood. The spots may appear different from those shown with the UV light. They should also
be circled. A comparison of the two visualization techniques can be made and may help in

5
identifying the unknown spots. The iodine method must be done after the UV method because the
compound may react with the iodine, possibly changing the results.

Calculate the Rf values for the known compounds and for all components of the analgesic. From
your results, identify which components are present in a particular analgesic. Some of the
components may have very similar Rf's under these conditions. However, their spots may look
different upon treatment with iodine and this may allow a distinction to be made. Note that caffeine
is present in small amounts and may not show up well. Purposely overloading a spot may help to
show a component present in small amounts.

You may choose from the following analgesics: Anacin, Extra Strength Excedrin, Extra Strength
Tylenol, and CVS Super Strength Pain Reliever. You may also analyze your personally-preferred
legal analgesic if you wish. Just bring a tablet to lab.

(2.) Solvent Effect on Rf Values. Three known compounds, anthracene, benzil, and
triphenylmethanol, will be developed in two different solvents. Prepare two identical plates, spotted
with each of those compounds, and develop one in ethyl acetate and the other in a mixture of 95%
hexane and 5% t-butyl methyl ether (you may use a second chamber for this solvent mixture).
Calculate Rf values, and in the post-lab write-up, discuss the effect of solvent on Rf, keeping in
mind the structures and polarities of the compounds and solvents.

OH
O

Anthracene Benzil Triphenylmethanol

SAFETY: Carefully note the warning about glass slivers above. Do not stare into the UV lamp.

BEFORE YOU LEAVE THE LAB: turn off any electrical equipment that you may have used,
put away your equipment and lock your drawer, clean up your work areas, close the fume
hood sash completely, and ask your TA for her or his signature. In general, please try to keep
the lab in as good condition as you found it. If you see caps off of bottles, replace the caps. If
you see spilled chemicals, clean them up or at least report it to your TA.

WASTE: Never dispose of glass waste in the regular trash. The custodian could become injured.
Dispose of all glass waste in the cardboard "Glass Only" boxes. Used capillary pipets may be placed
into those boxes also or into the dishes placed around the lab for that purpose. Dispose of all
solutions and developing solvents in the ORGANIC LIQUID WASTE container in the hood. When
finished, pour as much of the solvent as possible into the waste container and leave the screw-
capped jars open in the hood so they will dry. Leave the filter paper in the jars.

6
Postlab Questions

1.) Arrange the following compounds in order of increasing Rf in a TLC analysis: benzoic acid,
benzaldehyde, 3-heptanone, nonane, and cyclohexanol.

2.) Why must the spot be applied to the TLC plate above the level of development solvent?

3.) What will be the result of adding too much sample to the TLC plate?

4.) In a TLC analysis of analgesics, what would be the result if a solvent of too low polarity is used
to develop the plate?

5.) The Rf of ibuprofen was found to be 0.32 when t-butyl methyl ether was used as the
development solvent. What effect would there be on the Rf of ibuprofen if acetone had been used
to develop the TLC plate?

7
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Circular Dichroism & Optical Rotatory Dispersion

Polysaccharides Proteins
DNA
(agarose) (KCsa)

CHEM 305 Many biomolecules are α-helical!

How can we measure the amount and changes in amount of helical structure
of a biomolecule in solution?

Can we use x-rays?

need a crystal!

Can we use radiowaves (i.e. NMR)?

can’t measure changes

very readily

Can we use polarized light?

CHEM 305
Plane polarized light

Circularly polarized light

Take two polarized waves: one along x and the other along y, but with a phase
difference of λ/4

CHEM 305 Ex = E0 sin(2πz/λ) Ey = E0 cos(2πz/λ)

 clockwise or left-handed

from [Link]
CHEM 305
- Right-handed or anticlockwise circularly polarized light is obtained by using
Ey = E0 cos(2πz/λ)

- Combining left and right circularly polarized waves of equal amplitudes results
in polarized light.

-Combining left and right circularly polarized waves of unequal amplitudes

results in elliptically polarized light.

CHEM 305 [Link]

 Circular dichroism

We pass either left-handed or right-handed

light through an “optically active” substance.

What we find is that the amount of light

absorbed by the substance depends on
whether the light is left-handed or right-handed
circularly polarized.

Circular dichroism, at a given wavelength λ,

is defined as:

∆ε = εL – εR

where ε is the extinction coefficient. A

molecule is “optically active” if it is
chiral or asymmetric.

CHEM 305
Since the left- and right-handed light will be absorbed
differently, we will have ellipticity. The occurrence of
ellipticity is called circular dichroism.

An absorption band can be characterized by its rotational strength, which is

given by
R = (2.303)(3000)hc
32π3NA
∫ ∆ελ dλ
CHEM 305
where h is Planck’s constant and c is the speed of light.
Circular dichroism is observed only for wavelengths where the substance
absorbs light. For all other wavelengths, we will have optical rotation.

Optical rotation occurs as a consequence of a different refractive index for

left and right circularly polarized light components.

angle of rotation

incident light emergent light

CHEM 305
Optical rotation or circular birefringence results from different indices of
refraction for right- and left-circularly polarized light. The angle of rotation
depends on the nature of the substance, the thickness of the sample d,
and the concentration C, such that

α = [α] dC

where [α] is the specific rotation. We can measure a change in the optical
rotation with wavelength. This is known as optical rotatory dispersion (ORD).

CD

CHEM 305 ORD

 These hypothetical CD and
ORD spectra illustrate a
number of things:

1) Cotton effect – which is a

change in sign in the sign of
rotation.

How does this arise? ORD is

analogous to refractive index
so let’s take another look at
refractive index.

CHEM 305
induced dipole oscillation
What we need to determine now is how this induced dipole oscillation is
affected by changes in wavelength. To do this, let us perform the following
thought experiment:

record rotates slower than the ladybird

(long wavelength)

record rotates at same speed as the speed

of the ladybird
(resonance)

record rotates faster than the ladybird

(short wavelength)

CHEM 305
 rotation or λ

The same holds of the optical rotation or refractive index.

2) The quantity used to describe optical rotatory dispersion is not the specific
rotation [α] but rather the molar rotation [m’]:

[m’] = [α] 3 M
n2 + 2 100

where n is the refractive index of the medium and M is the molecular weight of
the solute. The term (3/n2 +2) is a correction factor which
accounts for the slight polarizability of the medium and its effect
CHEM 305 on the solute.
The previous equation relates the molar rotation to the specific optical rotation.
What is also needed is the relationship between [m’] and wavelength:

[m’] = 96π NA R λ02

hc λ2 –λ02
where R is the rotational strength, h is Planck’s constant, c is the speed of light,
and λ0 is the wavelength of the band. This equation is the Drude equation and
describes the response of an undamped harmonic oscillator driven by a periodic
force.

Putting λ = λ0, we see that this equation predicts that [m’] Æ ∞. This is not what
is observed experimentally. To get the correct description, a damping term must
be included.

CHEM 305
3) CD and ORD are related since both rely on the same underlying phenomenon.
They are related by the Kronig-Kramer relations. For example,
∞

[m’] = (2.303) 9000

π2
∫
0
∆ελ’ λ’2
λ2 – λ’2
dλ’

So why have techniques been developed to measure both CD and ORD?

CHEM 305
- Circular dichroism is an absorptive quantity and ORD is dispersive.
CD is a higher resolution method since we measure (relatively narrow)
absorption bands. In ORD, the dispersive peak is quite spread out –
if two bands are close to each other then it is difficult to distinguish them.

- ORD measurements can be performed at wavelengths where the substance

being investigated does not necessarily absorb light.

CHEM 305
Molecular basis for rotatory power

We have introduced the concept that circularly polarized light is absorbed

differently if it is left- or right-handed (CD) or that light is refracted differently
through a sample depending on whether it is left- or right-handed (ORD).
But why should this be? What is it about the substances that results in this
effect?

Coronene Hexahelicene

[α] = 3640o

CHEM 305
 Left-handed µ Right-handed
m

Right-handed
- The electric field will induce an oscillating dipole µ which will have a component
along the helix axis (recall the example on benzene shown earlier).
- Electric field is always accompanied by a magnetic field.
- An oscillating magnetic field parallel m to the helix axis will induce current in the
helix.
CHEM 305
The magnetic field for circularly polarized light has a component which is
parallel to the electric field. This is 90 degrees out of phase with the electric
field parallel to the helix axis. But its derivative will be in phase.

Therefore both the electric field and the magnetic field will contribute to an
electron displacement along a helical path. So we will have an electric and
a magnetic dipole moment.

Now if the circularly polarized light is

right-handed, the phase of the light
and that of the electron are the same.
In other words, both the electric field
and the magnetic field act in concert on
the electron.
µ m
If the circularly polarized light is left-handed,
then there is a phase difference of 180
degrees. In this case, the electric and
Right-handed magnetic fields acting on the electron
are in opposite directions.

CHEM 305
A consequence of this is that for a left-handed helix, the same arguments
would apply – but to the opposite handedness, i.e. left-handed circularly
polarized light would result in both the electric field and magnetic field
acting in concert on the electron.

Therefore a left-handed helix should display optical rotatory effects which

are just the opposite to those given by a right-handed helix.

One enantiomer of hexahelicene is optically active.

What about a racemic mixture?

CHEM 305
Previously we defined R, which is a rotational strength or optical rotatory power.
It can be shown (quantum mechanically) that R is

R = Im (µ · m)

i.e. the imaginary component of the scalar product between the electric and
magnetic moments. For most purposes, we can say that

R = µm cos θ

where θ is the angle between the two dipoles.

R is positive R=0 R is negative

CHEM 305
CHEM 305 - main application of CD
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 THE JOURNAL OF BIOLOGICAL CHEM~TRY
Vol. 242, No. 22, Issue of November 25, pp. 5336-5343, 1967
Pritied in U.S.A.

On the Mechanism of the Biosynthesis of

Prostaglandins E, and FIp*
(Received for publication, June 26, 1967)

MATS HAMBERG AND BENGT SAMUELSSON

From the Department of Chemistry, Karolinska In&it&et, and the Department of Medical Chemistry, Royal
Veterinary College, Stockholm, Sweden

SUMMARY The PGE and PGF compoundsare not interconvertible in the

The mechanismof the conversion of 8,11,14-eicosatrienoic system obtained from sheep vesicular gland* or guinea pig lung

Downloaded from [Link] by guest on November 8, 2020

acid into prostaglandin E1 (PGEl) and prostaglandin F1, (5).
Mechanistic studies have shown that the hydrogens at C-8,
(PGFI,) has been studied. Incubation of [13~-~H,3-“C]-
and [13~-3H,3J*C]8,11,14-eicosatrienoic acids showed that C-11, and C-12 of 8,ll ,lCeicosatrienoic acid remain in their
the hydrogen removed from C-13 during the conversion into original positions during the conversion to PGE1 (9) and that
PGEl and PGFI, has the L configuration. The conversion the 3 oxygen atoms of PGEl are derived from molecular oxygen
of [13~-3H,3J4C]8, 11,14-eicosatrienoic acid into PGEl is (10, 11). Furthermore, studies from this laboratory have
shown that the 2 oxygens of the 5-membered ring of PGEl
accompanied by a hydrogen isotope effect. No conversion
are derived from the same molecule of oxygen (11). Possible
of [2 J*C]lSL-hydroperoxy -8(cis), ll(cis) ,13(trans) - eicosa-
trienoic acid or [2-‘*C]lSL-hydroxy-8(cis), ll(cis) , lJ(frans) - mechanisms for the conversion have been proposed on the
basis of these experiments (11). The present paper provides
eicosatrienoic acid into PGEl or PGFI, could be detected.
Incubations of [9-3H,3J4C]8,11 ,14-eicosatrienoic acid re- additional information on the mechanism of the biosynthesis
vealed that PGF1, is not formed via PGEl in the system used. of prostaglandins, and especially on the initial step of the trans-
The mechanistic implications of the results obtained are formation.
discussed. It is suggested that 11-peroxy-8,12,14-eicosa-
MATERIALS AND METHODS
trienoic acid is the first intermediate in the conversion. The
peroxy acid is cyclized into an endoperoxide, which is even- Preparation of Stereospecifically Tritium-labeled Acids
tually transformed into PGEl or PGF,, by independent reac-
tions. Preparation of [11~-3H]Stearic Acid-Resolution of the cin-
chonidine salt of 3-hydroxydecanoic acid according to Cart-
wright (12) followed by regeneration of the acid and crystal-
lization from pentane yielded 3n-hydroxydecanoic acid, m.p.
4647” (reported, 46-47” (12)) ; [cr]: - 17.8” (c, 6.5, in chloroform)
(reported [a]:: -17.5” (12)). A solution of 184 mg of 3n-hy-
An enzyme system present in the vesicular gland of sheep
droxydecanoic acid in 3 ml of acetyl chloride was refluxed for
catalyzes the conversion of certain unsaturated fatty acids
15 min. The acid chloride formed was hydrolyzed by treatment
into prostaglandins (l-4). In whole homogenates of sheep
with 21 ml of 57% aqueous acetone at room temperature for 18
vesicular gland, most part of the product consists of prosta-
hours. After evaporation at a reduced pressure, 252 mg of
glandins of the E type. In whole homogenates of guinea pig
crude 3n-acetoxydecanoic acid were obtained. This material
lung, however, comparable amounts of PG&I and PGF2, are
was dissolved in methanol, and 2.2 g of monomethyl sebacate
formed from arachidonic acid (5). The formation of about
and 24 mg of sodium were added. A current of 0.1 ampere
equal amounts of PGE and PGF compounds in preparations
was passed through the solution for 14 hours. The solution
from the vesicular gland of sheep has also been reported (6-8).
was extracted with ether after addition of aqueous sodium
* This work was supported by a grant from the Swedish Medical bicarbonate to pH 10. The material present in the ether extract
Research Council (Projects 13X-217 and 13X-220). was hydrolyzed by treatment with 10% KOH in 50% aqueous
1 The abbreviations used are: PGEI, prostaglandin El, llc~,15-
methanol and was subsequently subjected to silicic acid chro-
dihydroxy-9-ketoprost-13-enoic acid; PGEI, &,15-dihydroxy-
9-ketoprosta-5,13-dienoic acid; PGFI,, prostaglandin FI,, 9a, matography. Crude lln-hydroxystearic acid was eluted with
lla,l5-trihydroxyprost-13-enoic acid; PGFz,, prostaglandin FtO, ether-hexane, 20:80. After esterification with methanol and
9a, IL, 15-trihydroxyprosta-5,13-dienoic acid; PGFIB, prostag- hydrochloric acid, the material was again subjected to silicic
landin F1#, 98, lh, 15-trihydroxyprost-l3-enoic acid; PGB,, 15-
hydroxy-9-ketoprosta-8 (12)) 13-dienoic acid. * M. Hamberg and B. Samuelsson, unpublished observations.

5336
Issue of November 25, 1967 M. Hamberg and B. Samuehm 5337

acid chromatography. Material eluted with ether-hexane n Ii OAC

‘q# ) OH 1) ACETVLATION ‘%#,H
(15:85), 149 mg, was crystallized from pentane, yielding methyl /c\ b /\
1 In-hydroxystearate (m.p. 50”). This compound was chemi- (CH~)‘ C”1 9 ANODIC COUP- (WI 1‘ (CH3),
LING WITH
CC, c’oon
cally pure as judged by gas chromatography. The identity cbocn,
HOOC -(CH1 1, - COOCH,
of the compound with methyl lln-hydroxystearate is based on
(a) the method of preparation; (b) chromatographic behavior on

1
30-hydrory- 1) on-
decanoic acid 2) CHINt
silicic acid chromatography and gas chromatography (retention [cd,“: -17, 5*( reported-17.5.) 3) 1s Cl
time on 1 y0 SE-30 equal to C-20.0; cj Reference 13) ; (c) melting (Cp6.5. chloroform)
point (reported by Bergstrom et al., 49.6498” (14), reported
by Tomecko and Adams, 4950” (15)), and (d) mass spectro-
metric analysis of the derivative obtained on chromic acid 1
oxidation. The mass spectrum showed ions of high intensity
+oc2 1)LiAl k
/\ A
at m/e 281 (ill - 31), 228 (M - 84; /3 cleavage with loss of y2 1. (yH*L 21cr0,
CH2=CH-(CHJ3--CHa), 213 (Al - 99; ar cleavage with loss bi, toon
of .(CHa)e-CHJ, 196 (M - 116; 228 - 32), 181 (M - 131;
[I1 L-‘Ii] STEARIC
213 - 32), 171 (Al - 141; @ cleavage with loss of .C&-C-

I
ACID 1) ACO-
2) otl-
II 3) CH,Nl
0 4) TSCI

(CHt)G---CHa), 170 (Ji - 142; fi cleavage with loss of CH,=C--

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I
OH Ii I) Li Al ‘H,
+,/ T 4
(CHZ)6CH3), and 142 (M - 170; p cleavage with loss of (CH< 14 ’ (Cl+,),
2) Cr 0,
CH2=CH-(CH2)c-COOCH3). This fragmentation pattern I
CH, ~0Oi-l
is in complete accord with that expected for methyl ll-keto-
stearate (cj. Reference 16). (HO-‘H] STEARIC
ACID
The preparation of [llnJH] stearic acid from methyl ll~-
hydroxystearate was carried out essentially as described by FIQ. 1. Reactions used for the preparations of [ll~-*HI- and
Schroepfer and Bloch (17). Methyl 1 In-hydroxystearate, [llL-%]stearic acids. Y’s, p-toluenesulfonyl.

1.5 mg, was dissolved in 2 ml of pyridine, and 200 mg of p-

toluenesulfonyl chloride were added after the solution had been fatty acid fraction obtained on hydrolysis with 10yO aqueous-
cooled to -15”. The solution was allowed to come to +4”, methanolic KOH for 2 hours under an atmosphere of argon was
and was kept at +4” for 150 hours. Extraction of the solution esterified by treatment with diazomethane and subjected to
with ether yielded the crude tosylate of methyl lln-hydroxy- chromatography on silicic acid impregnated with 257, AgNOa
stearate. This compound was purified by silicic acid chromatog- as the adsorbent. Elution of the column with ether-hexane
raphy (the tosylate was eluted with ether-hexane, 10:90). (20:80) yielded methyl [ll~-~H,l-i~C] 6,9,12-octadecatrienoate
The tosylate was dissolved in 5 ml of dry (distilled from LiAlHJ (15% yield based on incubated [ll~-~H,l-r~C] stearic acid).
tetrahydrofuran and was refluxed with 4 mg of LiA13H4 (10 mC, Chemical and radiochemical purity was demonstrated by gas-
obtained from New England Nuclear) for 20 hours. Extraction liquid radiochromatography with 8% ethylene glycol succinate
with ether yielded [l,ll~JH] stearyl alcohol. The stearyl S-X as the stationary phase. The isolated ester was diluted
alcohol was treated with 10% methanolic KOH for 1 hour at with 100 mg of unlabeled material and was elongated by means
reflux temperature in order to remove possible traces of readily of a malonic ester synthesis, performed as described by Klenk
exchangeable tritium. The stearyl alcohol was then dissolved and Mohrhauer (18). This procedure yielded [13~-3H,3-W]
in 1 ml of glacial acetic acid, and 1 ml of 95y0 acetic acid con- 8,11,14-eicosatrienoic acid in 20% yield. As judged from gas
taining 24 mg of CrO, was added. After 1 hour at 50”, meth- chromatography, the purity was at least 96%. The specific
anol was added and the crude stearic acid was extracted with activity was 0.16 PC of 3H and 0.05 PC of r4C per Mmole. Fig. 2
ether. After purification by means of silicic acid chromatog- summarizes the preparation of [13~-~H,3-~~C]8,11, lPeicosa-
raphy, 1 mg of [ll~-~H] stearic acid could be obtained. The trienoic acid.
compound was chemically and radiochemically pure as judged Preparation of [110-~11] Steuric d&d-The tosylate of methyl
by gas-liquid radiochromatography. The specific activity lln-hydroxystearate, 25 mg, was treated with 5.8 ml of glacial
was 105 PC per pmole. Fig. 1 summarizes the preparation of acetic acid containing 41.2 mg of sodium acetate at 60” for 24
[11 L-~H] stearic acid. hours (17). The crude methyl llcacetosystearate formed was
Preparation of [ISL-~H,S-‘~C] 8,ll ,l &Eicosatrienoic d cid-Af- hydrolyzed by treatment with 25% KOH in aqueous methanol.
ter addition of [l-W] stearic acid, the stereospecifically labeled Esterification by treatment with diazomethane followed by
stearic acid obtained as described above was incubated with a purification by silicic acid chromatography and crystallization
culture of Tetrahymena pyrijormis. The labeled stearic acid, from pentane yielded 6 mg of methyl lln-hydroxystearate
2 mg, was dissolved in 1 ml of ethanol and added to 200 ml of a (m.p. 48”). No impurities could be detected by gas chroma-
medium consisting of 0.5% glucose, 0.5% Difco yeast extract, tography. Preparation of [ll~-~H] stearic acid from the ob-
and 0.5% Pepton aus Fleisch (E. Merck) in 0.004 M phosphate tained methyl lln-hydroxystearate was carried out by the
buffer pH 7.0. After 42 hours at 30”, the cells were collected procedures described above for the preparation of [ll~-~H]
by centrifugation of the culture at 1000 x g for 20 min. The stearic acid from methyl lln-hydroxystearate. After purifica-
5338 Biosynthesis of Prostaglandins Vol. 242, No. 22

partition chromatography, [9L-3H] palmitic acid (5% yield)

[ll~-~ii] OCTADECANOIC
ACID
was obtained. The purity was established by gas-liquid chro-
matography on 1% SE-30. The specific activity was 0.9 PC
per pmole.
Preparation of Methyl Palmitoleate--The two palmitic acids
obtained as described above were mixed with [l-‘“Cl palmitic
acid and were added to growing cultures of Corynebacterium
diphtheriae, strain CT s( -) tox( -), kindly provided by Pro-
E,~-~ti,,-‘~Cj 6,9,12-OCTA- fessor A. M. Pappenheimer. The medium described by Mueller
DECATRIENOIC ACID
and Miller (20) was used after supplemention with tryptophan,
calcium pantothenate, and biotin (21). The cultures were
incubated for 24 hours at 36” on a rotating shaker. The cells

1
Malonic
l 2c ester were collected by centrifugation of the cultures at 800 x g
synthesis and were treated with 20% KOH in 50% aqueous methanol for
2 hours at reflux temperature. The saponifiable material was
treated with diazomethane and applied to a column of silicic
acid containing 35% AgN03. Methyl palmitoleate and methyl
h3L-‘Ii, 3-q 8,11,14-
oleate were obtained by elution with ether-hexane, 10:90. No
EICOSATRIENOIC ACID
traces of methyl palmitate could be detected in this material
FIG. 2. Preparation of [~~L-~H,~-I~C]S, 11,14-eicosatrienoic acid by gas-liquid chromatography with 10% ethylene glycol suc-

Downloaded from [Link] by guest on November 8, 2020

from [ll~-~H] stearic acid. The same reactions were used for the
cinate S-X on Gas-Chrom P. In a separate experiment with
preparatiou of [131~-~H,3-‘~C]S,ll, 14-eicosatrienoic acid from
[llo-VI]stearic acid. [1J4C] palmitic acid, the obtained monoenoic ester fraction
was analyzed by gas-liquid radiochromatography on 10%
tion by silicic acid chromatography, 5 mg of [ll~-~H] stearic ethylene glycol succinate S-X. The peak of radioactivity co-
acid were obtained. The compound was chemically and radio- incided with the mass peak of methyl palmitoleate. No ra-
chemically pure according to gas-liquid radiochromatography. dioactivity was associated with the mass peak of methyl ole-
The specific activity was 17 FC per pmole. Fig. 1 summarizes ate.
the preparation of [ll~-~H] stearic acid. The percentage retentions of tritium of the methyl palmit-
Preparation of [lS~-~H,S-~~C]8,11,14-Eicosatrienoic Acid- oleates relative to the incubated palmitic acids are given in
Methyl [lln-3H,1-14C]6,9,12-octadecatrienoate isolated after Table I (methyl palmitoleate derived from methyl 6,9,12-
incubation of [ll~-~H,l-‘~C] stearic acid with T. pyriformis octadecatrienoate formed from [l ~LJH, 1-“Cl-stearic acid,
(10% yield) was diluted with unlabeled material and then Experiment I; methyl palmitoleate derived from [ll~-~H]
elongated by malonic ester synthesis. The obtained [13~-~H, stearic acid, Experiment II).
3-W]8,11,14-eicosatrienoic acid (37 % yield) was at least 95 % Preparation of Methyl 9-Hydroxypelargonate-Part of the
pure as judged from gas chromatography. The specific ac- obtained methyl palmitoleates was dissolved in 2 ml of chloro-
tivity was 0.11 PC of 3H and 0.03 PC of 14C per pmole. form and treated with ozone at -15” for 5 min. After 10 min
at room temperature, 2 ml of 50% aqueous ethanol containing
Localization of Tritium Label of
270 mg of NaI%H4 were added. The solution was stirred vig-
6,9,l%Octadecatrienoic Acid
orously for 10 min at 0” and then for 2 hours at, room temperature.
In order to determine whether the tritium label of 6,9,12- The material obtained by extraction with chloroform was
octadecatrienoic acid formed by enzymatic desaturation of subjected to silicic acid chromatography. The radioactive
the stereospecifically tritium-labeled stearic acid remained in material was eluted with ether-hexane, 30:70. Part of this
its original position and retained its original configuration, the material was treated with 50 ~1 of pyridine, 15 ~1 of hexameth-
following experiments were carried out.
Preparation of Palmitic rlcid-Experiment I : Methyl 6,9,12- TABLE I
octadecatrienoate formed from [ll~-~H,l-~~C] stearic acid was Relention of b-ilium in mcth?/l palmitoleales, methyl
hydrogenated and saponified. This material, diluted with 9-hydroxypelargonates, and dimelhyl azzlales
unlabeled stearic acid (10 mg, 6.6 PC of 3H), was dissolved in formed from palmilic acids
20 ml of acetone, and 200 mg of KMn04 were added (19). After In Experiment I, palmitic acid was derived from methyl F,9,12-
20 hours at 40”, water, NaHS03 solution, and 2 N hydrochloric octadecat,rienoate formed from [ll~-~H,1-~43] stearic acid. In
acid were added. Material isolated by extraction with ether Experimeut. II [9L-aH] palmitic acid was formed from [ll~-~H]
was subjected to reversed phase partition chromatography with stearic acid.
the following solvent systems: methanol-water, 216 ml:84 ml
Retention of tritium
(moving phase) ; 2,2,4+imethylpentane, 30 ml (stationary Compound
phase). Palmitic acid could be isolated in 4% yield. The pur- Experiment I Experiment II
ity of the compound was checked by gas-liquid chromatography
% %
with 1% SE-30 on Gas-Chrom P. The specific activity was 0.2
$2 per pmole. Palmitic acid.. 100 100
Methyl palmitoleate. 93 91
Experiment II: A sample of [ll~-~H] stearic acid was diluted 90 88
Methyl 9-hydroxype1argonat.e.
with unlabeled stearic acid and subjected to oxidation with Ijimethyl azelat,e. 7 3
KMn04 in acetone as described above. After reversed phase
Issue of November 25, 1967 M. Hamberg and B. Samuelssoln

yldisilazane, and 5 ~1 of trimethylchlorosilane for 30 min at

room temperature. The retention time of the main peak on
15% silicone grease corresponded to C-12.6 (cj. Reference
22).
Preparation of Dimethyl Azelate-The methyl 9-hydroxy- OSN N*Otlin
‘Hl”C : 1.51 ( 100% ) %i/“C i 1.59 ( 99% )

pelargonate obtained as described above was dissolved in 2 ml

of acetic acid, and 2 ml of 95$‘& acetic acid containing 20 mg of
CrOs were added. After 30 min at 37”, methanol was added,
and the solution was extracted with ether. Material obtained
after evaporation of the ether was treated with diazomethane
and subjected to silicic acid chromatography. The radioactive
material was eluted with ether-hexane, 10:99. The retention
time on 15% silicone grease corresponded to C-12.0 (cf. Reference
22). The relative retentions of tritium of the dimethyl azelates
and methyl 9-hydroxypelargonates are given in Table I.
As shown in Table I, methyl palmitoleate formed from [9caH] ‘H/‘~c:0.07(4%) ~n/‘~c=1.59(99%1

palmitic acid (Experiment II) retained most of the 3H label, II I

indicating that the 9L hydrogen is retained during the desatura- FIQ. 3. Reactionsusedfor the localization of the tritium label
tion. This is in agreement with the findings of Schroepfer and of PGEl biosyuthesizedfrom [13~-‘B,3-“C]8,11,14eicosatrienoic
Bloch (17) that the hydrogens of the L configuration are retained acid.

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and the hydrogens of the D configuration are lost from carbon
atoms 9 and 10 during the conversion of stearic acid to oleic 100

acid by C. diphtherial, strain C s( -) tox( -). Methyl pal- 90

mitoleate formed from palmitic acid derived from methyl 6,9, 90

t
12-octadecatrienoate (Experiment I) also retained most of the 70

aH label, showing that the precursor was [9GaH] palmitic acid. s

60

The findings that the methyl 9-hydroxypelargonates retained P

: 50.
the tritium label whereas the dimethyl azelates lost the tritium +
c
40
label show independently that the aH label was at C-9. The ;
:

-$ 30.
fact that [9caH] palmitic acid was formed from methyl 6,9,12-
octadecatrienoate derived from [ll~-aH,1-14C] stearic acid in-
dicates that no changes in position or configuration of the 3H
label occurs during the transformations of the stereospecifically 90 100 120 1&O ‘60 I90 100 220m,~‘0 260 290 300 320 3‘0 360 390
tritium-labeled stearic acids into 6,9,12-octadecatrienoic acids
by T. pyn’formis. Fm. 4. Mass spectrum recorded on the trimethylsilyl ether
derivative (TMSi) of CompoundI.
Localization of Tritium Label at C-13 of PGE,
The reactions shown in Fig. 3 were used to localize the tritium after evaporation of the chloroform was subjected to silicic
label of PGE, biosynthesized from [13~-~H ,3J4C]8,11, 14- acid chromatography. Elution with ethyl acetate-benzene,
eicosatrienoic acid. PGE, formed on incubation of the pre- 20:80, yielded CompoundI (Fig. 3). Part of the material was
cursor with the 866 x g supernatant of a homogenate of sheep converted into its trimethylsilyl ether by treatment with a
vesicular gland (71% conversion) was isolated by silicic acid mixture of 59 ~1 of pyridine, 15 ~1 of hexamethyldisilazane,and
chromatography. 5 ~1 of trimethylchlorosilane for 30 min at room temperature.
Preparation of Compound I (Fig. S)-Treatment of PGEi The derivative was subjected to gas-liquid chromatography
obtained as described above with NaBHa in methanol solution with 150/, siliconegreaseon 196 to 129 meshCelite. A single
yielded a mixture of PGFr, and PGFia. PGFi, was isolated peak with a retention time equal to C-26.6 (cf. Reference22)
by reversed phase partition chromatography with Solvent appearedon the gaschromatogram. The purity of CompoundI
System C-38 (23) and was treated with diazomethane and was also establishedby thin layer chromatography. Only one
acetylated. After purification by silicic acid chromatography spot appeared (RF 0.24). The massspectrum of the trimeth-
(the methyl ester and triacetate of PGFr, were eluted with ylsilyl ether derivative (Fig. 4) showedions of high intensity
ethyl acetate-benzene, 10:96), the derivative was dissolved in at m/e 355 (M - 75; lossof CHaOH + CHaCO.), 313(M - 117;
2 ml of chloroform and cooled to -15”. A stream of ozone was 10~s of Cl&OH + CH&O* + CHzCO), 310 (M - 120; 10s~ of
passed through the solution for 4 min at -15”. After 10 min 2 CHaCOOH), 295 (M - 135; 10~s of CH&OOH + CHaOH +
at room temperature, 2 ml of 59% aqueous ethanol containing CHaCO.), 221 (M - 209; 10s~ of 2 CHaCOOH + -OTMSi),
270 mg of NaBHl were added. The mixture was stirred vig- 267 (M - 223; lossof 2 CHaCOOH + .CHsOTMSi) and 167
orously for 10 min at 0” and then for 2 hours at room temperature. (M - 263; 10~s of 2 CHaCOOH + *(CHa)sCOOCHa),thus con-
After dilution with water, 6 N hydrochloric acid was added firming the identity of the obtained compoundwith Compound
dropwise until the pH reached 1. The chloroform layer was I. The procedure used for reductive ozonolysis was a slight
separated, and the aqueous phase was extracted three times modification of the proceduregiven by Sousaand Bluhm (24).
with chloroform. The chloroform phases were combined and Preparation of CompoundZZ (Fig. S)-To a solution of Com-
washed with water until neutral reaction. The residue obtained pound I in 1 ml of acetic acid, 1 ml of 95% acetic acid containing
5340 Biosynthesis of Prostaglandins Vol. 242, No. 22

100 were sprayed with rhodamine, and the spots were visualized
90 under ultraviolet light.
60
Gas-&i&id Chromatography-Gas-liquid chromatography was
performed with an F and M Biomedical gas chromatograph,
s 70 model 400. The stationary phasesused were silicone grease
h 60
.Z (15%, supported on 100 to 120 mesh Celite), SE-30 (1% on
s 50 Gas-Chrom P), and ethylene glycol succinateS-X (8% on Gas-
E
:
40 Chrom P).
-
; 30 Measurement of Radioaetitity-For the measurement of
radioactivity, the methodsdescribedin an accompanyingreport
20
(25) were used.
10
Preparationof [W4c] I&-Hydroperoxy-8(&s) ,ll (cis), lS(trans)-
60 100 I20 140 160 160 200 220m,e240 260 280 3M) 320 340 360
eicosatrienoic Acid-A solution of the ammoniumsalt of [2J4C]*
8,11,14-eicosatrienoicacid wasprepared by dissolving4 mg of
FIG. 5. Maas spectrum recorded for Compound II the fatty acid in 1 ml of 0.019 M NH’OH. After cooling to
0”, 2.5 ml of borate buffer, pH 9.0, containing 2.5 mg of soybean
10 mg of CrOa was added. After 30 min at 37”, methanol was lipoxidase (20,000 units per mg, purchasedfrom Fluka) were
added, and the solution w&s extracted with ether. The residue added. After 15 min at 0”, an additional 0.5 ml of buffer con-
obtained after evaporation of the ether w&s treated with di- taining 0.5 mg of lipoxidase was added. The incubation was
azomethane and subjected to preparative thin layer chroma- interrupted after 30 min at 0” by the addition of 15 ml of ethanol.

Downloaded from [Link] by guest on November 8, 2020

tography. Material forming the main spot (RF 0.48) was The solution was immediately diluted with water, acidified to
eluted from the silica gel with methanol and was subjected to pH 3, and extracted with ether three times. The ether was
silicic acid chromatography. Elution with ethyl acetate- washeduntil neutral reaction and wasevaporated under reduced
benzene (10:90) yielded Compound II. Part of the material pressureat room temperature. Purification by silicic acid chro-
was subjected to gas-liquid chromatography with 15% silicone matography yielded [2J4C] 15chydroperoxy-S(ci.s),ll(cis) ,
grease on 100 to 120 mesh Celite. A single peak with a retention 13(tran.s)-eicosatrienoic
acid (cf. Reference 25). The material
time equal to C-20.5 (cf. Reference 22) appeared on the gas was more than 90% pure as judged from thin layer chromatog-
chromatogram. The mass spectrum recorded on the compound raphy.
(Fig. 5) showed ions of high intensity at m/e 284 (M - 102; Preparation of [dJ4C]16L-Hydroxy-8(cis),ll(cis),lS(trans)-
loss of CHsCOOH + CHICO), 252 (M - 134; loss of [Link] Acid-Sodium borohydride, 20 mg, was added to
CHaCOOH + CH,CO + CHsOH), 234 (M - 152; loss of 2 a solution of 5 mg of [2-14C]15L-hydroperoxy-8(cis),ll(ci.s),
CH&OOH + CHSOH), 225 (M - 161; loss of CH&OOH + 13(trans)-eicosatrienoicacid in 1 ml of methanol. After 20
CH&OO . + CH,CO), 224 (M - 162; loss of 2 CHsCOOH + min at 0” and 40 min at room temperature, the product was
CH&O), 220 (M - 166; loss of CH&OOH + C&CO + 2 extracted with ether and then subjectedto silicic acid chroma-
CHsOH), 206 (M - 180; loss of 3 CH&OOH), and 202 (M - tography. The [2-W]15L-hydroxy-8(cis),ll (cis),l3(trans)-eicos-
184; loss of 2 CHaCOOH + 2 CHsOH), thus confirming the atrienoic acid obtained was more than 90% pure as judged by
identity of the compound with Compound II. thin layer chromatography.
Preparation of [9-3H,3-14c]8,11 ,lJ-Eicosatrienoic Acid and
Methods [ll -3H,W4c]8,11,I,$-Eicosatrienoic Acid-The preparation of
Preparation of Homogenates-Homogenatesof sheep ve- [9-3H,3J4C]8,11,14-eicosatrienoicacid is described in detail
sicular glands,29% wet weight to volume, were prepared in a in an accompanyingreport (26). The sameprocedurewasused
modified Biicher medium. The supernatant obtained after for the preparation of [l lJH, 2J4C]8,11,lCeicosatrienoic
centrifugation at 800 x g for 20 min wasusedfor the incubations. acid.
Incubations were also performed in a system consisting of
RESULTS
microsomes,high speedsupernatant, and tetrahydrofolate (6).
Incubations and Treatments--The incubations were inter- Incubation of [130-3H,3-14C]-and [13dH,3-14C]8,11,14-
rupted after 60 min at 37” by the addition of 7 to 10 volumesof Eicosatrienoic A&&--The 3H: 14Cratios and the percentage
ethanol. After precipitated protein w&9 removed by filtration, retentions of tritium of PGEl biosynthesized from the two
dilution with water, and acidification, the solution waa ex- stereospecificallylabeled precursorsare given in Fig. 6. The
tracted three times with ether. The ether phase w&s washed tritium label is retainedduring the conversionof [13~2H, 3-*‘Cl*
until neutral reaction and w&sevaporatedunder reducedpressure. 8,11,1Ceicosatrienoic acid into PGE1. That the tritium
The residue was subjected to silicic acid chromatography. remainsat C-13 wasshownby degradationexperiments. PGEl
Silicic Acid Chromatography-Silicic acid chromatography formed from [13~-~H ,3-14C]8,11,14-eicosatrienoic acid was
was carried out on silicic acid (Mallinckrodt, 100 mesh) ac- reducedwith NaBH4, and PGR, wasisolated. The derivative
tivated at 115’. The columns were eluted with increasing formed by treatment with diazomethaneand acetylation was
concentrations of ethyl acetate in benzene or with increasing subjected to reductive ozonolysis. Compound I (Fig. 3) was
concentrationsof ether in hexane. isolated and was found to retain the tritium label (99% re-
Thin Layer Chromatography-Thin layer chromatography tention relative to PGE1). However, on oxidation of Compound
was carried out on Silica Gel G. The moving phasewas the I to II, the label was lost (4% retention of tritium relative to
organic layer of the following solvent system: ethyl ace- PGEJ . The finding that PGBl (15-hydroxy-9-ketoprosta-
tate-2,2,4-trimethylpentane-water, 75:75: 100. The plates 8(12), 13dienoic acid) formed from PGEl by treatment with
 Issue of November 25, 1967 M. Hamberg and B. Samuelsson 5311

0.5 N NaOH retained the tritium label (98% retention relative

to PGEJ was independent evidence that no tritium label was
present at C-12 or C-8 of PGE1. During the conversion of
[13QH, 3-14C]8, 11,lPeicosatrienoic acid to PGE1, most of the
tritium label is lost. PGR, was found to retain the tritium
label when biosynthesised from [~SD-~H, 3J4C]8, 11,14-eicos-
atrienoic acid (88% retention), but to lose the tritium label
when formed from [ 13~-~H, 3-14C]8, 11,lCeicosatrienoic acid
(7 % retention). These experiments therefore indicate that
the removal of hydrogen from C-13 of 8,ll,lPeicosatrienoic
15 hydroperoxy -
acid is stereospecific. The hydrogen removed has the L con- 8.11.13 - cicosalrtenoic 8.11.13-cicosalr~cnoic
figuration. acid scld
Furthermore, the 8,11,14-eicosatrienoic acid recovered after coon

incubation of [ 13~-~H, 3-14C]8, 11, ICeicosatrienoic acid was

enriched in tritium. After 750/, conversion the retention of
3H was 284%, and after 64y0 conversion, 226%. Thus the
removal of the 13~-~H is accompanied by isotope discrimination PGE,

which is of the same order of magnitude as that recently found FIQ. 7. Incubations of [2-‘%]15L-hydroperoxy-S(cis), 11 (cis) ,-
in the conversion of [13~-~H, 3J4C]8, 11,14-eicosatrienoic acid 13(lrans) eicosatrienoic acid and [2-%]15L-hydroxy-8(&), -
into [3-14C]15L-hydroperoxy-8(cis), ll(ci.s) ,13(truns) -eicosatri- ll(cis),l3(trans)-eicosatrienoic acid with homogenates of sheep

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enoic acid catalyzed by soybean lipoxidase. The retention of vesicular gland.
3H was, after 59% conversion, 20297,; after 69% conversion,
253%; and after 81% conversion, 341% (25). TABLE II
zncubation of [2-‘v]15L-HydrOpeTOxy-8(Ci.s) ) 11 (cis) ,ld(truns)- Results obtained on incubation of [9-3H,3-14C]8,11 ,l&eicosatrienoic
eicosatrienoic Acid and [W4CJ15~-Hydroxy-S(cis) , 11 (cis) , IS acid and (11 -3H,S-14C]8,11 ,14-eicosatrienoic acid with whole
(trans)-eicosatriencoic Acid-The products obtained after incuba- homogenates (I) and washed microsomes and SOY0 of original
amount of high speed supernatant plus 1.5 M excess
tion of [2-14C]15L-hydroperoxy-8(cis), ll(ci.s) ,13(truns)-eicosatri-
of tetrahydrofolate (II) (26)
enoic acid (1 mg) and [2J4C]15L-hydroxy-8(ciS) , 11 (cis) ,13
PGE, and PGF,a were isolated as methyl esters by thin layer
(truns)-eicosatrienoic acid (1 mg) with the 800 X g supernatants
chromatography with Solvent System MI (27)
obtained from 5 g of vesicular glands were subjected to silicic
acid chromatography. No radioactive PGEl or PGFI, could Retention of :H
by products
be detected (Fig. 7). In both cases, only one peak of radioac- Incubation
system
tivity appeared on silicic acid chromatography. This material PGEl PGFL,
had the same chromatographic behavior as that of 15L-hydroxy-
8(&s), ll(ci.s) ,13(trans)-eicosatrienoic acid. The incubated % %
[2-“C]15~ - hydroperoxy -8(&s), 11 (cis) ,13(truns) - eicosatrienoic [9-aH,3-W]8, 11,14-Eicosatrienoic
acid was apparently reduced to the hydroxy acid during the acid... .._.............. I 3
[9-3H,3-1*C]8,11,14-Eicosatrienoic
incubation. The reduction might well have been nonenzymatic,
acid......................... II 24 136
since the hydroperoxy acid also was reduced to the hydroxy acid
[11-3H,2J4C]8, 11,14-Eicosatrienoic
when incubated with boiled homogenates of sheep vesicular acid.............. .._.... I 97
gland. The reduction also took place when the hydroperoxy [ll-3H,2-~4C]8,11,14-Eicosatrienoic
acid............................ II 84 112

,i3L-% , 3-‘q 9.11.14-

- “G” I-In
acid was treated at 0” with a solution of reduced glutathione
in Biicher’s medium; however, similar treatment with TPNH
-EICOSATRIENOIC ACID did not give any reduction.
INCUBATED ACID, ‘H/‘)C:1.36 (100%) PGE,
Zncubation of [9-sH , P4C]8, 11,l .@icos&ienoic Acid and
RECOVERED ACID.‘H/‘k=3.67 (264%)
‘H/“C.O.O7 (5%) [f 1 -3H, I-14C’j8, 1 1 , 1 Q-Eicosatrienoic A cd-The percentage re-
(AFTER 15% CONVERSION)
tentions of tritium of PGEl and PGFl, biosynthesized from
[9-3H, 3-14C]- and [l 1-3H, 2J4C]8, 11,14-eicosatrienoic acids are
given in Table II.
CO” -
DISCUSSION

fj3D-‘H , 3-‘&d 6.11.14- It has been shown earlier that the 2 oxygen atoms of the 5-
~I~~sATRIENOIC ACID membered ring of PGEl originate in the same molecule of oxygen
INCUBATED ACID, +I/% = 1.61 (100%) PGE,
(11). The fact that neither 15L-hydroperoxy-8(ci.s), 11 (cis) ,13
‘n/“C= 1.61 ( 99%) (tram)-eicosatrienoic acid nor 15L-hydroxy-8(&s), ll(czL?),l3
(tram)-eicosatrienoic acid is converted to PGEl indicates that
FIQ. 6. Biosynthesis of PGEl from stereospecifically [13-JH]-
labeled precursors. The acids, 1 mg, were incubated with the the initial step in the conversion involves a dioxygenase reaction
800 X g supernatants obtained from 5 g of sheep vesicular glands. in which oxygen is introduced at C-11 or simultaneously at
5342 Biosynthesis of Prostaglandins Vol. 242, No. 22

ELIMINATION OF The oxygenation at C-11 and C-9 was also studied by using
precursors labeled with tritium at C-11 and C-9. As expected,
aoH 5
PGEl for&ed by incubation of [9-3H, 3-14C]8, 11,14-eicosatri-
enoic acid with whole homogenates lost most of the tritium
~3L-%-3%J- 8.1114-
label, and PGEl formed by incubation of [ll-3H, 2J4C]8, 11,14-
EICOSATRIENOIC ACID
eicosatrienoic acid with whole homogenates retained most of
L
L 4
the tritium label (Table II).
microsomes and supernatant
In a system consisting of washed
plus tetrahydrofolate, about equal
amounts of PGEl and PGR, were formed from 8,11, lCeicos-
atrienoic acid (6, 26). As shown in Table II, PGFI, biosyn-
thesized from [9-3H, 3-14C]8, 11,lCeicosatrienoic acid in this
system retained the tritium label and was in fact enriched with
respect to tritium relative to the precursor. The fact that
PGF1, formed from [9-3H, 3J4C]8, 11,14-eicosatrienoic acid
FIG. 8. Two hypothetical pathways in t,he biosynthesis of PGEl retained the 3H label excludes the possibility that PGEl is an
intermediate in the formation of PGF,, in this system. The
enrichment of tritium in PGF1, supports the contention that
the endoperoxide discussed above is the last intermediate com-
mon to both PGEl and PGFI,. The transformation of this
intermediate into PGEl involves abstraction of the hydrogen

Downloaded from [Link] by guest on November 8, 2020

at C-9, which apparently proceeds with isotope discrimination.
The intermediate, which in this way becomes enriched with
respect to tritium, is converted to PGFI, without elimination
of tritium. The tritium enrichment therefore appears in
PGF1,.
The PGEl fraction biosynthesized from [9-3H, 3J4C]8, 11,14-
eicosatrienoic acid in the presence of washed microsomes and

HO *
w
I 2RH2

COOH
supernatant plus tetrahydrofolate had a higher retention
3H than PGE, formed from the same precursor with the use of
of

whole homogenate. One possible explanation for this finding

is that a compound isomeric with PGEl (9,15-dihydroxy-ll-
ketoprost-13-enoic acid) is formed when the system consisting
FIG. 9. Mechanism proposed for the conversion of 8,11,14- of washed microsomes, supernatant, and tetrahydrofolate is
eicosatrienoic acid to PGEl and PGFI,. used. The formation of this compound from 8,ll ,lCeicos-
atrienoic acid has also been suggested by Nugteren, Beerthuis,
C-11 and C-9 of the precursor acid. Thus, two different mech- and van Dorp (7). Support for the formation of an isomer of
anisms for the incorporation of the oxygens of the ring seemed PGEl with the use of washed microsomes is furnished by the
feasible (11). This is shown in Fig. 8. One of the pathways finding that the PGEl fraction formed from [11-3H,2-14C].
shown involves addition of 1 molecule of oxygen with concom- 8,ll,lCeicosatrienoic acid in the system containing washed
itant formation of the bond between C-8 and C-12. In the microsomes, supernatant, and tetrahydrofolate had a lower
other pathway, 11-peroxy-8,12,14-eicosatrienoic acid is formed retention of tritium than did PGEl formed from the same pre-
in a lipoxidase type of reaction. In the formation of this com- cursor with the use of whole homogenate (Table II). The
pound, 1 hydrogen is lost from C-13 in the first step, whereas slight enrichment of tritium found in PGFI, biosynthesized from
the first pathway involves loss of the hydrogen at a later state. [ll-3H ,2J4C]8, 11,14-eicosatrienoic acid is also in agreement
The absolute configuration of the hydrogen removed from C-13 with the formation of 9,15-dihydroxy-11-ketoprost-13-enoic
of 8,11,14-eicosatrienoic acid during the conversion into PGE1 acid (the isotope discrimination accompanying the conversion
(and PGF1,) was determined by incubation of [13~-~H,3-1~C]- of the endoperoxide into 9,15-dihydroxy-11-ketoprost-13-enoic
and 13~-~H, 3J4C]8, 11,14-eicosatrienoic acids. These experi- acid appears in PGFI,). The fact that PGF1, formed from this
ments showed that the hydrogen of the L configuration was precursor retains the tritium label indicates that no 11-keto
selectively removed from C-13. The finding that the conversion compound is formed in the biosynthesis of PGF1, and is in
of [13~-3H, 3-14C]8, 11,14-eicosatrienoic acid proceeds with agreement with its formation from the endoperoxide.
enrichment of tritium in the precursor indicates that a reaction
of the lipoxidase type is involved in the initial transformation. Acknowledgments-We wish to thank Mr. U. Inger for his
In the next step, 11-peroxy-8,12,lCeicosatrienoic acid is cy- skillful technical assistance during the preparation of the ster-
clized into the endoperoxide shown in Fig. 9. This reaction eospecifically tritium-labeled acids. We also thank Miss G.
involves attack by oxygen at C-15, isomerization of the Al2 Borg for her capable technical assistance.
double bond into the Al3 position, formation of the bond between
REFERENCES
C-8 and C-12, and formation of an endoperoxide. The en-
1. BERGSTROM, S., DANIELSSON, H., AND SAMUELSSON, B., Bio-
doperoxide is finally transformed to PGEl by removal of the chi,m. Biophys. Acta, 90, 207 (1964).
hydrogen at C-9, or into PGFI, by reductive opening of the 2. VANDORP, D.A., BEERTHUIS, R.K., NUGTEREN, D.H., AND
peroxide. VONKEMAN, H., Biochim. Biophys. Acta, 90, 204 (1964).
Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

1. SUMMARY OF TEST PRINCIPLES AND CLINICAL RELEVANCE

A. Total Cholesterol
Cholesterol is measured enzymatically in serum or plasma in a series of coupled reactions that
hydrolyze cholesteryl esters and oxidize the 3-OH group of cholesterol. One of the reaction
byproducts, H2O2 is measured quantitatively in a peroxidase catalyzed reaction that produces a
color. Absorbance is measured at 500 nm. The color intensity is proportional to cholesterol
concentration. The reaction sequence is as follows:

cholesteryl ester hydrolase

Cholesteryl ester + H2O ------------------------------------->cholesterol + fatty acid

cholesterol oxidase
Cholesterol + O2 ----------------------------> cholest-4-en-3-one + H2O2

peroxidase
2H2O2 + 4-aminophenazone + phenol ---------------------> 4-(p-benzoquinone-
monoimino)-phenazone + 4 H2O

Elevated levels of cholesterol increase the risk for coronary heart disease (CHD). Cholesterol is
measured to help assess the patient's risk status and to follow the progress of patient's treatment
to lower serum cholesterol concentrations. Desirable cholesterol levels are considered to be
those below 200 mg/dL in adults and below 170 mg/dL in children.

B. Triglycerides

Triglycerides are measured enzymatically in serum or plasma using a series of coupled reactions
in which triglycerides are hydrolyzed to produce glycerol. Glycerol is then oxidized using glycerol
oxidase, and H2O2, one of the reaction products, is measured as described above for cholesterol.
Absorbance is measured at 500 nm. The reaction sequence is as follows:

lipase
Triglycerides + 3H2O ------------> glycerol + fatty acids

glycerokinase
Glycerol + ATP ----------------------> glycerol-3-phosphate + ADP

glycerophosphate oxidase
Glycerol-3-phosphate + O2 ------------------------------> dihydroxyacetone phosphate + H2O2

peroxidase
H2O2 + 4-aminophenazone + 4-chlorophenol -------------> 4-(p-benzoquinone-monoimino)-
phenazone + 2H2O + HCl.

High levels of serum triglycerides help mark conditions that are associated with increased risk for
CHD and peripheral atherosclerosis. High triglycerides are associated with increased risk for
CAD in patients with other risk factors, such as low HDL-cholesterol, some patient groups with
elevated apolipoprotein B concentrations, and patients with forms of LDL that may be particularly
atherogenic. Desirable fasting triglyceride levels are considered to be those below 200 mg/dL,
and are further categorized as Borderline, 200-400 mg/dL; High, 400-1,000 mg/dL; and Very High
(> 1000 mg/dL). Very high triglycerides can result in pancreatitis and should be promptly
evaluated and treated. Triglycerides are also measured because the value is used to calculate
low density lipoprotein (LDL)-cholesterol concentrations (see below). In NHANES 2003-2004,

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Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

triglycerides are only measured in specimens from fasting participants, i.e., those sampled in
Session 1.

C. High density lipoprotein (HDL) cholesterol

Low serum concentrations of HDL-cholesterol are associated with increased risk for CHD.
Coronary risk increases markedly as the HDL concentration decreases from 40- to 30 mg/dL. A
low HDL-cholesterol concentration is considered to be a value below 35 mg/dL, and high HDL,
>60 mg/dL. HDL-cholesterol values are also used in the calculation of LDL-cholesterol (see LDL
section below).

Direct HDL method. HDL is measured directly in serum. The basic principle of the method is as
follows. The apoB containing lipoproteins in the specimen are reacted with a blocking reagent
that renders them non-reactive with the enzymatic cholesterol reagent under conditions of the
assay. The apoB containing lipoproteins are thus effectively excluded from the assay and only
HDL-chol is detected under the assay conditions.

The reagents are purchased from Roche/Boehringer-Mannheim Diagnostics. The method uses
sulfated alpha-cyclodextrin in the presence of Mg+2, which forms complexes with apoB containing
lipoproteins, and polyethylene glycol-coupled cholesteryl esterase and cholesterol oxidase for the
HDL-cholesterol measurement. The reactions are as follows:

(1) ApoB containing lipoproteins + α-cyclodextrin + Mg+2 + dextran SO4 ---> soluble non-reactive
complexes with apoB-containing lipoproteins
PEG-cholesteryl esterase
(2) HDL-cholesteryl esters > HDL-unesterified cholesterol + fatty acid

(3) Unesterified chol + O2 PEG-cholesterol oxidase > cholestenone + H2O2

(4) H2O2 + 5-aminophenazone + N-ethyl-N-(3-methylphenyl)-N’_succinyl ethylene diamine

+ H2O + H+ peroxidase > qunoneimine dye + H2O

Absorbance is measured at 600 nm.

D. LDL-cholesterol

Most of the circulating cholesterol is found in three major lipoprotein fractions: very low density
lipoproteins (VLDL), LDL and HDL.
[Total chol] = [VLDL-chol] + [LDL-chol] + [HDL-chol]

LDL-cholesterol is calculated from measured values of total cholesterol, triglycerides and HDL-
cholesterol according to the relationship:
[LDL-chol] = [total chol] - [HDL-chol] - [TG]/5
where [TG]/5 is an estimate of VLDL-cholesterol and all values are expressed in mg/dL.

LDL carries most of the circulating cholesterol in man and when elevated contributes to the
development of coronary atherosclerosis. LDL-cholesterol is measured to assess risk for CHD
and to follow the progress of patients being treated to lower LDL-cholesterol concentrations.
Desirable levels of LDL-chol are those below 130 mg/dL in adults and 110 mg/dL in children. In
NHANES 2001-2002, LDL-chol will be reported only for fasting participants >5 years of age.

2. SAFETY PRECAUTIONS

A. Daily Safety Precautions. All personnel working in the laboratory must wear gloves and
laboratory coats. Laboratory coats are to be kept buttoned. Gloves are removed when leaving
the immediate work area or when entering offices within the immediate work area. All used

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Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

gloves, vials, pipettes and other items that come in contact with specimens are disposed of in a
Biohazard box lined with a red plastic bag. Such biohazardous waste is picked up each day by
housekeeping for proper disposal. Work benches are cleaned at the end of each day with a
solution of sodium hypochlorite (bleach:water, 1:100, v/v) and then covered with plastic-backed
white paper.

B. Blood Handling. The improper handling of blood samples from patients with infectious diseases,
e.g., hepatitis or HIV, can lead to infection of staff that draw, handle, analyze or store such
samples. Transmission can occur by ingestion, inhalation or direct contact, and staff must
exercise care when handling blood samples. Always wear liquid impermeable gloves (e.g., latex
or plastic) when handling biological samples. Never pipet samples by mouth. Avoid contact with
serum. Cover any scratches or cuts on fingers and hands and wear gloves before handling
serum. Store all samples in sealed containers. In order to minimize the formation aerosols, do
not leave samples open to the atmosphere longer than necessary.

It is about 30 times easier to become infected with hepatitis than with HIV through sample
mishandling, and it has been recommended that the usual precautions for handling blood
specimens to prevent hepatitis infection serve as a guide to prevent AIDS infection as well.
Handle all specimens as if you know them to be infectious. All staff should adhere to the CDC
Guidelines for Prevention of HIV Infection in Health Care Workers.

C Spills. The contaminated area is cleaned with a solution of sodium hypochlorite (bleach:water, 1
:100, v/v) and the wipes are disposed of in a red biohazard bag.

3. COMPUTERIZATION; DATA SYSTEM MANAGEMENT

Sample identifying information is received via a closed E-mail system from the NCHS data repository
(WESTAT) the same day the samples are received in the laboratory from the MEC. The E-mail
sample information is downloaded into a working laboratory database (referred to here as the
NHRDR database) and computerized run lists are created for the automated analyzer. When an
analytical run is completed, the analytical data are downloaded from the analyzer in the form of ASCII
text files, which are then converted to a database and uploaded to the NHRDR database. When all
specimen analyses are completed and reviewed, a transmittal file is prepared and sent by E-mail to
WESTAT in the form of a comma delimited file. The quality control data are also transmitted by E-
mail at the same time.

The file structures for the electronic shipment log files are shown on the next two pages. The first
table indicates general information about the laboratory, address, name of shipment file, and other
information. The second table, labeled 'SEND FILE' illustrates the structure of the electronic
shipment list transmitted to the laboratory with each batch of specimens. This file is used to import
sample information into the laboratory databases.

The third table, labeled 'RESULTS FILE: Lipids-Vessel ID 21' shows the format of the transmittal file
prepared by the laboratory to transmit results to WESTAT. The first 7 lines in the table contain the
information from the 'SEND FILE.' Note that while the data in the tables on the next two pages break
the information down by data field for the sake of clarity, all the data for each NHANES 2001-2002
sample-person is transmitted in a single comma delimited record.

The fourth table, is labeled 'QC FILE: Lipid-Vessel ID 21' and illustrates the structure of the quality
control transmittal files. Again, all information for each analytical run is transmitted in a single record.

Occasionally, a specimen may have a value considered to be a critical, or “panic value”. A panic
value is a value that can reflect a life-threatening problem if not attended to promptly. Of the analytes
measured by the NHANES 2001-2002 lipid laboratory, the only one for which such a panic value
must be considered is triglyceride. Extremely high triglyceride levels (i.e., levels > 2,000 mg/dL) can

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Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

result in pancreatitis, a potentially life threatening condition. For this reason, the triglyceride panic
value for NHANES 2001-2002 is set at 1,000 mg/dL. Values of 1,000 mg/dL or higher are
communicated to NCHS by fax or telephone as soon as they are detected. (See section 13).

4. SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES; CRITERIA FOR

SPECIMEN REJECTION

A. General. Various factors can affect lipid and lipoprotein measurements.

(1) Fasting. Recent food intake exerts little effect on plasma total cholesterol concentration.
Plasma triglycerides, however, increase in postprandial plasma to an extent that is related to
the fasting triglyceride levels and the amount of fat intake. This is due to the appearance of
chylomicrons in the circulation after a fat-containing meal. Chylomicrons are normally
cleared within 9-12 hr, and no chylomicrons should be present after a 12 hr period of fasting.
Transient decreases in HDL-chol and LDL-chol also occur, the magnitude of which depends
on the fat content of the meal. In NHANES 2001-2002, triglyceride is measured only in
specimens drawn from participants who have fasted at least 9 hours before venipuncture.

(2) Serum vs. plasma. In general, anticoagulants exert osmotic effects in which water leaves the
cells and enters the plasma, thus diluting the plasma and lowering the concentrations of non-
diffusible components. The magnitude of this effect depends on the anticoagulant used and
its concentration. Serum cholesterol and triglyceride concentrations are about 3-5% higher in
serum than in EDTA plasma, although no significant serum-plasma difference was observed
for HDL. Thus, the serum concentrations of lipids and lipoproteins probably reflect more
accurately the subjects' physiological state at the time of venipuncture. Serum is used for
measuring lipids and lipoproteins in NHANES 2001-2002, as it has been for previous HANES
surveys.

(3) Sample volumes. The sample volumes required are as follows: total cholesterol and/or
triglyceride, 0.5 ml; HDL measured with the direct method, along with total cholesterol, 0.2 ml.
Any sample remaining after analyses are complete are returned to -80 oC, and subsequently
sent to the NHANES 2001-2002 serum bank as directed by NCHS.

(4) Serum shipment and storage container. In NHANES 2001-2002, plastic, screw top cryovials
are used to ship and store serum. Different size vials are used for 3-5 year old children and
those >5 years old.

(5). Storage and sample stability. Serum can be stored at -20°C in a non-self defrosting freezer
for up to 4 weeks. For longer storage (> 4 weeks) they should be maintained at -80°C or
lower. Total cholesterol, triglyceride and HDL-cholesterol are stable for at least one year at
-80 oC or lower.

B. Specimen handling

(1) Collect blood into a glass tube such as a red top Vacutainer® blood collection tube.

(2) Allow the blood to stand for 45 min at room temperature to allow complete clotting and clot
retraction. A shorter period may result in incomplete clotting and secondary clots may form
later. During the clotting period leave the collection tube sealed.

(3) Centrifuge the samples at 1,500 x g for 30 min at 4°C. It is preferable to use a refrigerated
centrifuge for this purpose, but an unrefrigerated centrifuge can be used if necessary. In
either case, the samples should be placed into an ice bath immediately after centrifuging and
maintained at 2-4° C thereafter.

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Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

(4) Samples should be kept frozen at -20°C, in a non-self defrosting freezer until shipped to the
laboratory. If a shipment must be delayed longer than 4 weeks, the specimens should be
kept at -80° C. In the event a shipment may have been thawed and refrozen prior to
shipment, this should be noted on the transmittal form.

(5) Samples are shipped by overnight carrier, such as Federal Express. Samples are not
shipped on Friday or the day before a holiday, since the laboratory is closed on weekends or
holidays. NCHS provided lists of shipment dates that take account of the weekend and
holiday schedule. However, in the event it becomes necessary for the laboratory to receive a
shipment on a weekend or holiday, NCHS will inform the laboratory of this, and the laboratory
makes arrangements to receive the shipment.

5. PROCEDURES FOR MICROSCOPIC EXAMINATIONS; CRITERIA FOR REJECTION OF

INADEQUATELY PREPARED SLIDES

Not applicable for this laboratory and procedures specified.

6. EQUIPMENT AND INSTRUMENTATION, MATERIALS, REAGENT PREPARATION, CALIBRATORS

(STANDARDS), AND CONTROLS

Cholesterol, triglyceride and HDL-cholesterol analyses are performed on a Hitachi 704 Analyzer
which is serviced by Roche Diagnostics (formerly Boehringer-Mannheim Diagnostics), Indianapolis,
IN. Cholesterol is measured enzymatically using the Cholesterol High Performance reagent ([Link].
704036), Roche Diagnostics). Triglycerides are analyzed enzymatically simultaneously with
cholesterol using reagents from the same manufacturer (Triglycerides/GPO, cat. no. 1488872).
Triglyceride blanks are measured in CDC surveillance materials using the same reagent, but without
lipase. Direct HDL-cholesterol reagent is obtained from Roche Diagnostics (Direct HDL, cat. no.
1661442), and analyzed simultaneously with cholesterol and triglycerides. If for some reason,
analyses must be delayed, the specimens should be kept frozen at -80° C until they are analyzed.

Specimens to be analyzed for cholesterol, triglyceride and HDL-cholesterol can be stored for up to 1
year at -80° C.

A. Reagents required to operate instrument.

(1) Cell Clean 90, cat no. 1224310, Roche Diagnostics. (This is a solution of NaOH, concentration
not specified, used to keep reaction cells free of protein deposits). Store at room temperature
until expiration date indicated for the lot.

(2) Hitergent, cat no. 409149, Roche Diagnostics. pH 12.5, solution contains 5% ethanolamine, an
unidentified antibacterial agent and an unidentified non-ionic detergent. Store at 10-35 oC until
expiration date indicated for the lot.

(3) Working solution, 2% Hitergent. Add 20 mL Hitergent to deionized water and bring to 1,000
mL. Store at room temperature for up to 4 months.

B. Test specific reagents

(1) Cholesterol Reagent: The components of Cholesterol High Performance System Pack
Reagents (Roche Diagnostics, Indianapolis, IN) include (taken from package insert):
Cholesterol Reagent (16 x 50 mL)
75 mmol/L PIPES buffer, pH 6.8
10 mmol/L Mg2+
0.2 mmol/L Sodium cholate

Page 7 of 23
Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

0.15 mmol/L 4-Aminophenazone

> 4.2 mmol/L Phenol
> 0.5 U/mL Cholesterol esterase (EC [Link]; pseudomonas species; 25° C)
> 0.15 U/mL Cholesterol oxidase (EC [Link]; E. coli; 25° C)
> 0.25 U/mL Peroxidase (EC [Link]; horseradish; 25° C)
1% Fatty alcohol-polyglycol ether
Buffer, unspecified stabilizers, unspecified preservative

The reagent is supplied as a solution and is ready to use. After being opened, the reagent is
stable for 28 days at 2-12 °C, or 7 days at room temperature. Protect reagent from light.

(2) Triglyceride Reagents: The components of the Triglycerides (GPO) System Pack include (from
package insert):

50 mmol/L PIPES buffer, pH 6.8

40 mmol/L Mg++
0.20 mmol/L Sodium cholate
> 1.4 mmol/L ATP
> 0.13 mmol/L 4-Aminophenazone
4.7 mmol/L 4-Chlorophenol
1 μmol/L Potassium hexacyanoferrate (II)
0.65% Fatty alcohol polyglycolether
> 5.0 U/mL lipoprotein lipase (EC [Link]; Pseudomonas species, 25oC)
> 0.19 U/mL glycerolkinase (EC [Link]; Bacillus stearotheromophilus; 25oC)
> 2.5 U/mL glycerophosphate oxidase (EC [Link]; E. coli; 25oC)
> 0.10 U/mL Peroxidase (EC [Link]; horseradish; 25°C)
unspecified preservative

The reagent is supplied as a solution and is ready for use. When opened, the solution is stable
for 14 days at 2-12° C, or 7 days at room temperature (15-25° C).

(3) Direct HDL-cholesterol method

(a) The Direct HDL-cholesterol reagents, R1 and R2 contain the following components (from
package insert):

R1 Cyclodextrin/Buffer, supplied as a solution, ready to use.

0.5 mmol/l α-cyclodextrin
0.5 g/l dextran sulfate
7.0 mg/ml magnesium sulfate (MgSO4)
0.3 g/l EMSE
10 mmol/l MOPS (3-morpholino-propane sulfonic acid) buffer, pH 7.0)
unspecified preservative

(b) R2 Buffer/PEG-enzyme/4-Aminophenazone, is supplied as a lyophilized mixture and is

reconstituted with diluent supplied in the reagent kit. R2 contains the following approximate
concentrations after reconstitution:

> 1 kU/l PEG cholesterol esterase (EC [Link]; Pseudomonas species; 25oC)
> 5.6 kU/l PEG cholesterol oxidase (EC [Link]; Pseudomonas species; 25oC)
> 30 kU/l peroxidase (EC [Link]; horseradish; 25oC)
0.5 g/l 4-aminophenazone
10 mmol/l MOPS (3-morpholino-propane sulfonic acid) buffer, pH 7.0
Detergent and preservative (unspecified)

(c) Preparation:

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Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

The R1 Reagent solution is ready for use. The R2 Reagent (bottle 2a) requires
reconstitution using the pre-measured contents of the diluent vial (bottle 2). The procedure
is as follows: Carefully pour about 1/2 of the diluent solution (bottle 2) into bottle 2a. Mix
gently until the lyophilized material is fully dissolved. Carefully pour the reagent solution
back into bottle 2, allowing bottle 2a to drain completely. This transfer should be done as
quantitatively as possible, but without rinsing bottle 2a after the transfer. Mix the solution
gently for a couple of minutes. The solution is now ready for use. R1 after opening, and
R2 after reconstitution are stable for 28 days at 2-12° C.

C. Storage and Stability:

(1) Cholesterol reagent. Store the open cholesterol System Pack Reagent on the analyzer at 2-
12° C. The solution is stable for 4 weeks at 2-12° C or 7 days at 20-25° C when protected
from light and contamination by microorganisms.

(2) Triglyceride reagent. Store triglyceride reagent on the analyzer at 2-12 oC. The reagent is
stable for 14 days at 2-12° C or 7 days at room temperature.

(3) Heparin. Store unopened heparin at 20-25° C. Stable until the expiration date printed on the
bottle label.

(4) Direct HDL-cholesterol. R1 Reagent is stable unopened up to the stated expiration date.
When open, R1 reagent is stable up to 28 days on the analyzer at 2-12° C. R2 Reagent is
stable up to the expiration date prior to reconstitution. After reconstitution, the R2 is stable up
to 28 days on the analyzer at 2-12° C.

D. Equipment

500 ul positive displacement pipettes (SMI, Inc.)

250 ul positive displacement pipettes (SMI, Inc.)
50 ul pipettes, (Absoluter)
1.4 mL microcentrifuge tubes
Wheaton Step-Pette TM

7. CALIBRATION AND CALIBRATION VERIFICATION PROCEDURES

The Hitachi analyzer is calibrated at the beginning of the week and as necessary thereafter. A one
point calibration procedure is used for total cholesterol, triglyceride and Direct HDL-cholesterol.
Frozen serum calibration pools (Soloman Park Laboratories, Kirkland, WA) are used to calibrate total
cholesterol and direct method HDL-cholesterol assays. Triglycerides are calibrated using calibration
sera (Precical, cat no 620213) obtained from the Roche Diagnostics.

A. Daily Check
The following procedures are performed at the beginning of each work day before the first
analytical run.
(1) Check water supply.
(2). Check 2% Hitergent supply**.
(3) Check Cell Clean 90 supply**.
(4) Prepare reagents, controls and calibrators as needed.
(5) Exchange incubation bath water: Press MAINTENANCE, then press 1 ENTER.
(6) Perform Photometer Check.
(7) Air purge: "Start Conditions" display.
(8) Wipe sample and reagent probes.
(9) Probe adjust.
** Refer to Section 6. Reagents, above

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Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

B. Full Calibration:

If the CRT display shown is in the Routine Job Menu, press NEXT or BACK to move to the
Calibrator & Control Test Selection display. If the CRT display shown is not in the Routine Job
Menu, press ROUTINE, then press 3 ENTER.

(1) PHASE: The Instrument stores operating parameters and calibration data for two sets of
twenty chemistries each. These two sets of chemistries are referred to as PHASE 1 and PHASE
2. The two phases are defined in the Channel Assignment display. To indicate which set of
chemistries is being used, type the phase number (1 or 2), the press ENTER. For NHANES
2001-2002, all tests are in the PHASE 1 set.

(2) CALIBRATION TYPE: Press 1 ENTER to specify "Start-Up" Calibration.

(3) STANDARD TYPE: Press 1 ENTER to select tests for the blank (saline). If test selection for
the blank is stored in memory, the tests in memory appear at the right margin of the display.

(4) TESTS: Activate the appropriate test or profile keys for those tests which require a Blank
Calibrator, then press ENTER. (Each test key is activated when its LED is illuminated). The tests
assigned appear at the right margin of the display, and the STANDARD TYPE entry field
displays: "STD 2-6."

(5) TESTS: Press the appropriate test keys for those tests which require a standard or
standards, then press ENTER. The tests assigned appear at the right margin of the display and
the STANDARD TYPE entry field displays: "ISE 1,2." Advance the cursor to the CALIB LOAD
LIST entry field. Press 1 ENTER.

(6) If you do not want to run controls, update the System Disk with calibrator test selection as
follows:
Advance the cursor to FD READ/WRITE.
Press 2 ENTER.
The CRT displays: "WRITE OK?"
Press 1 ENTER (YES).

Note: It is not absolutely necessary to write calibrator test selection data on the System Disk.
However, if the laboratory experiences a power failure, this step prevents permanent loss of test
selection information.

Wait while the System Disk is updated, then proceed to ROUTINE PATIENT TEST SELECTION.

C. Blank Calibration Only:

If the CRT display shown is in the Routine Job Menu, press NEXT or BACK to move to the
Calibrator & Control Test Selection display. If the CRT display shown is not in the Routine Job
Menu, press ROUTINE, then press 3 ENTER.

(1) PHASE: The instrument stores operating parameters and calibration data for two (2) sets of
twenty chemistries each. These two sets of chemistries are referred to as PHASE 1 and PHASE
2. The two phases are defined in the Channel Assignment display. For NHANES 2001-2002, all
tests are in the PHASE 1 set.

To indicate which set of chemistries is being used, type the phase number (1 or 2), then press
ENTER.

(2) CALIBRATION TYPE: Press 1 ENTER to specify "Start-Up" Calibration.

Page 10 of 23
Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

(3) STANDARD TYPE: Press 1 ENTER to select tests for the blank (saline) update. If previous
test selections for blanks are stored in memory, the tests in memory will appear at the right
margin of the display.

(4) TESTS: Activate the appropriate test or profile keys for those tests requiring a blank update,
then press ENTER. (Each test key is activated when its LED is illuminated.) The tests assigned
appear at the right margin of the display, and the STANDARD TYPE entry field displays: "STD 2-
6".

(5) TESTS: Deselect all previously selected tests so that no tests are selected for "STD 2-6",
then press ENTER. No tests should appear at the right margin of the display, and the
STANDARD TYPE entry field now displays: "ISE 1,2".

(6) Press ROUTINE, then press 4 ENTER and the Start Conditions screen will appear on the
display.

(7) Enter the START SAMPLE NO. and request START UP CALIBRATION. Verify that a control
interval of 1 or greater has been selected. All runs of 15 samples or more require 3 sets of
control pools per run. (Tests requiring controls were selected in Routine Job No. 3)

(8) Press START to begin the calibration.

D. Reagent/calibrator changes

The primary means of following any manufacturer initiated changes in reagent or calibrator
formulations is the laboratory quality control system. Such reagent or calibrator changes are
generally minor, if visible at all. However, they have the potential for introducing minor abrupt
shifts in the laboratory mean, which are normally detected through the QC system.

When the laboratory is informed of an impending change, the Laboratory Operations Coordinator
will take steps to procure the new reagent or calibrator in sufficient time to perform analyses in
parallel with the old and new reagent or calibrator. Such parallel analyses will be conducted over
a period of 3-4 weeks in 10 runs, each of which is performed on a different day. Each parallel run
will be performed on the same day, and will include the appropriate QC pools and at least 20
specimens, for a total of 200 specimens over the 10 runs. This is accomplished by setting up
additional instrument channels with the new reagent or calibrator and analyzing the sample
simultaneously in both the normal and new channels.

At the end of the 10 runs, descriptive statistics (mean (SD), %CV) will be determined for each
analyte in each QC pool and a paired t-test will be used to assess the significance of the
differences between like analytes in each pool. Descriptive statistics (mean (SD), paired t-tests,
and linear regression analyses relating the two arms of the parallel analyses will also be
conducted for the affected analytes in the 200 split specimens analyzed with the old and new
reagents or calibrators. These data will serve to characterize the effect of new reagent or
calibrator formulations on NHANES 2001-2002 analyses.

Note that the laboratory has no control over reagent or calibrator formulations, and will have to
use the new reagent if it replaces the current formulation. The data collected above, however,
will be useful during the data analysis phase when the desirability of adjusting NHANES 2001-
2002 data to account for such systematic bias changes can be considered. Based on past
experience, such systematic biases are expected to be minimal and would probably be
considered acceptable without adjustment. Nonetheless, the data will be available for such
adjustment should this not be the case.

Page 11 of 23
Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

17. TEST RESULT REPORTING SYSTEM; PROTOCOL FOR REPORTING CRITICAL CALLS (IF
APPLICABLE)

Test results are reported to the NCHS at a frequency and by a method determined by the study
coordinator. Generally, data from this analysis are compiled and sent to the responsible person at
the collaborating agency as an ASCII text file or Excel file, either through electronic mail or on a
diskette.

For NHANES 1999+, all data are reported electronically to the Westat ISIS computer and then are
transferred to NCHS.

18. TRANSFER OR REFERRAL OF SPECIMENS; PROCEDURES FOR SPECIMEN

ACCOUNTABILITY AND TRACKING

The Microsoft Access database is used to keep records and track specimens for NHANES.

19. SUMMARY STATISTICS AND QC GRAPHS

A. Total cholesterol

Summary Statistics for Total Cholesterol by Lot

Standard Coefficient
Lot N Start Date End Date Mean Deviation of Variation
SL2I132 218 1/15/2003 1/27/2005 165.2 2.3 1.4
SL3I180 218 1/15/2003 1/27/2005 244.2 2.8 1.2

2003-2004 Total Cholesteral Quality Control

300

SL3I180
250

200

SL2I132

150

100

50

0
1/15/2003 4/25/2003 8/3/2003 11/11/2003 2/19/2004 5/29/2004 9/6/2004 12/15/2004

Page 20 of 23
Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

B. Direct HDL

Summary Statistics for HDL-Cholesterol by Lot

Standard Coefficient
Start
Lot N Date End Date Mean Deviation of Variation
SL4I267 218 1/15/2003 1/27/2005 28.0 0.6 2.3
SL4I236 218 1/15/2003 1/27/2005 50.3 0.8 1.7

2003-2004 HDL-Cholesterol Quality Control

60

SL4I236

50

40

SL4I267
30

20

10

0
1/15/2003 4/25/2003 8/3/2003 11/11/2003 2/19/2004 5/29/2004 9/6/2004 12/15/2004

Page 21 of 23
Total Cholesterol, Direct HDL, Precipitated HDL, Triglycerides, and LDL
NHANES 2003-2004

C. Triglycerides

Summary Statistics for Triglycerides by Lot

Standard Coefficient
Lot N Start Date End Date Mean Deviation of Variation
SL2I132 218 1/16/2003 1/27/2005 86.7 1.8 2.1
SL3I180 218 1/16/2003 1/27/2005 205.6 3.1 1.5

2003-2004 Triglycerides Quality Control

250

SL3I180

200

150

100 SL2I132

50

0
1/16/2003 4/26/2003 8/4/2003 11/12/2003 2/20/2004 5/30/2004 9/7/2004 12/16/2004

Page 22 of 23